It has been suggested that CXCR3 is important for nociception. Our experiments were conducted to evaluate involvement of CXCR3 and its ligands (CXCL4, CXCL9, CXCL10, CXCL11/CCL21) in neuropathic pain. Our studies give new evidence that intrathecal administration of each CXCR3 ligand induces pain-like behaviour in naive mice that occurs shortly after injection due to its location of neurons, which is confirmed by immunofluorescent staining. Moreover, intrathecal administrations of CXCL9, CXCL10, CCL21 neutralizing antibodies diminished pain-related behaviour. RT-PCR/Western blot analysis unprecedentedly showed spinal elevated levels of CXCR3 after chronic constriction injury of the sciatic nerve in rats in parallel with different time-course changes of its endogenous ligands. Initially, on day 2 we observed spinal increased levels of CXCL10 and CXCL11 indicating that these chemokines have important roles in triggering neuropathy. Then, on day 7, we observed increased levels of CXCL4, CXCL9, CXCL10. Interestingly, changes in CXCL9 level persisted until day 28, suggesting that these chemokines are responsible for long-term, persistent neuropathy. Additionally, in DRG the CXCL4, CXCL9 were elevated. The results obtained from primary glial cultures, suggests that all CXCR3 ligands can be produced in microglia, but also, except for CXCL4, in astrocytes. We provide the first evidence that in neuropathy chronic intrathecal administration of CXCR3 antagonist, (±)-NBI-74330, attenuates hypersensitivity with concomitant occurrence of microglial and some of CXCR3 ligands activation observed in the spinal cord and/or DRG level. This paper underlies the significance of CXCR3 in neuropathic pain and shows therapeutic potential of its blockade for enhancement of morphine analgesia as the major novelty of this work.

Surgical stress has been shown to facilitate the tumor growth and metastasis of colon cancer. To unravel the mechanisms underlying surgery induced-colon cancer progression, a syngeneic transplantation tumor model was established with murine colon cancer CT26 cells and the effect of laparotomy on tumor progression was investigated. Especially the expression of several CXC chemokines was assayed, and its roles in regulating myeloid-derived suppressor cells (MDSCs) recruitment were analyzed. We found that laparotomy promoted in vivo tumor growth and angiogenesis. CXCL4 expression was significantly downregulated by laparotomy in the tumor tissue and the peritoneal cavity. Functionally, CXCL4 overexpression significantly reduces tumor volume compared to control. Through analysis of CD11b+/Gr1+ MDSCs cell, we found an upregulated proportion of MDSCs in the tumor tissues and peritoneal cavity following laparotomy, and this enhancement was blocked after CXCL4 overexpression. Further, a negative correlation was found between CXCL4 expression and MDSC amounts in clinical samples. Higher CXCL4 expression and lower MDSCs proportion is positively related to overall survival.

CONCLUSIONS

Surgical trauma contributes to colon cancer progression by downregulating CXCL4 and hence promoting MDSC recruitment, which leads to an immunosuppressive environment.

Papillary thyroid cancer (PTC) is the most prevalent endocrine neoplasia. The increased incidence of PTC in patients with thyroiditis and the frequent immune infiltrate found in PTC suggest that inflammation might be a risk factor for PTC development. The CXCR3-ligand system is involved in thyroid inflammation and CXCR3 has been found upregulated in many tumors, suggesting its pro-tumorigenic role under the inflammatory microenvironment. CXCR3 ligands (CXCL4, CXCL9, CXCL10 and CXCL11) trigger antagonistic responses partly due to the presence of two splice variants, CXCR3A and CXCR3B. Whereas CXCR3A promotes cell proliferation, CXCR3B induces apoptosis. However, the relation between CXCR3 variant expression with chronic inflammation and PTC development remains unknown. Here, we characterized the expression pattern of CXCR3 variants and their ligands in benign tumors and PTC. We found that CXCR3A and CXCL10 mRNA levels were increased in non-metastatic PTC when compared to non-neoplastic tissue. This increment was also observed in a PTC epithelial cell line (TPC-1). Although elevated protein levels of both isoforms were detected in benign and malignant tumors, the CXCR3A expression remained greater than CXCR3B and promoted proliferation in Nthy-ori-3-1 cells. In non-metastatic PTC, inflammation was conditioning for the CXCR3 ligands increased availability. Consistently, CXCL10 was strongly induced by interferon gamma in normal and tumor thyrocytes. Our results suggest that persistent inflammation upregulates CXCL10 expression favoring tumor development via enhanced CXCR3A-CXCL10 signaling. These findings may help to further understand the contribution of inflammation as a risk factor in PTC development and set the basis for potential therapeutic studies.

The term atherosclerosis refers to the condition of deposition of lipids and other substances in and on the artery walls, called as plaque that restricts the normal blood flow. The plaque may be stable or unstable in nature. Unstable plaque can burst and trigger clot formation adding further adversities. The process of plaque formation involves various stages including fatty streak, intermediate or fibro-fatty lesion and advanced lesion. The cells participating in the formation of atherosclerotic plaque include endothelial cells, vascular smooth muscle cells (VSMC), monocytes, monocytes derived macrophages, macrophages and dendritic cells and regulatory T cells (T_REG). The role of a variety of cytokines and chemokines have been studied which either help in progression of atherosclerotic plaque or vice versa. The cytokines involved in atherosclerotic plaque formation include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-20, IL-25, IL-27, IL-33, IL-37, TNF-α, TGF-β and IFN-γ; whereas amongst the chemokines (family of small cytokines) are CCL2, CCL3, CXCL4, CCL5, CXCL1, CX3CL1, CCL17, CXCL8, CXCL10, CCL20, CCL19 and CCL21 and macrophage migration-inhibitory factor. These are involved in the atherosclerosis advancements, whereas the chemokine CXCL12 is play atheroprotective roles. Apart this, contradictory functions have been documented for few other chemokines such as CXCL16. Since the cytokines and chemokines are amongst the key molecules involved in orchestrating the atherosclerosis advancements, targeting them might be an effective strategy to encumber the atherosclerotic progression. Blockage of cytokines and chemokines via the means of broad-spectrum inhibitors, neutralizing antibodies, usage of decoy receptors or RNA interference have been proved to be useful intervention against atherosclerosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the PMF pathogenesis have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic-stromal cross-talk remain elusive. Using an unbiased approach and validation in MPN patients, we identified that the differential spatial expression of the chemokine CXCL4/platelet-factor-4 (PF4) marks the progression of fibrosis. We demonstrate that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis and decreases 1) the activation of pro-fibrotic pathways in megakaryocytes, 2) inflammation in fibrosis-driving cells and 3) JAK/STAT activation in both megakaryocytes and stromal cells in three murine PMF models. Our data indicate that higher CXCL4 expression in MPN has pro-fibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.

Background: The presence of neutrophil-rich inflammation in colon tissues of patients with ulcerative colitis (UC) is one of the most important histological characteristics of this disease. However, the expression of CXCL chemokines governing the infiltration of neutrophils in UC has not been well elucidated. Materials and methods: In this experimental study, the UC model was induced in Wistar rats by administration of 2 mL 4% acetic acid into the large colon through the rectum. Animals were anesthetized after 48 hrs; their colon tissue samples were isolated for macroscopic and histopathological examinations. The expression of CXCL family was assessed by reverse transcription polymerase chain reaction (qRT-PCR) technique. Results: Heavy infiltration of neutrophils, coagulation necrosis, and ulcers were observed in H&E staining, which pathologically proved the UC model. qRT-PCR results showed that ELR⁺ CXC chemokines such as CXCL6 and CXCL3 had the highest expression in the UC group, which was 49 and 28 times higher than that of the control group, respectively. In addition, other chemokines of this group including CXCL1, CXCL2, and CXCL7 had a significant increase compared to the control group (P≤0.05). However, ELR^- CXC chemokines such as CXCL4, CXCL13, and CXCL16 showed a smaller upregulation, while CXCL14 chemokine showed a significant decrease compared to the control group (P≤0.05). However, the expression of CXCL9-12 and CXCL17 did not change. Conclusion: The results showed that the ELR⁺ CXC chemokines, especially CXCL6 and CXCL3, many involved in the pathogenesis of UC; therefore, CXCL6 and CXCL3 chemokines can be used as therapeutic targets for UC, although more studies using human samples are required.

The management of advanced ovarian cancer is challenging due to the high frequency of recurrence, often associated with the development of resistance to platinum‑based chemotherapy. Molecular analyses revealed the complexity of ovarian cancer with particular emphasis on the immune system, which may contribute to disease progression and response to treatment. Cytokines and chemokines mediate the cross‑talk between cancer and immune cells, and therefore, present as potential biomarkers, reflecting the tumor microenvironment. A panel of circulating C‑C motif chemokine ligand (CCL) and C‑X‑C motif chemokine ligand (CXCL) chemokines were examined in the serum of 40 high‑grade patients with ovarian cancer prior to primary surgery. The level of immune infiltration in tumors was also analyzed. The preoperative levels of chemokines differ between patients. Elevated levels of circulating CXCL4 + CCL20 + CXCL1 combination can discriminate patients with shorter recurrence‑free survival and overall survival. The presence of tumor‑infiltrating T lymphocytes was detected in half of the patients. The mRNA expression analysis suggests the presence of antitumoral and immunosuppressive elements in the tumor microenvironment. The combination of circulating CXCL9 + CXCL10 can distinguish immune‑infiltrated tumors that will lead to shorter recurrence‑free survival. The results suggest that preoperative profiling of circulating chemokines in patients with ovarian cancer may provide valuable information regarding tumor recurrence and immune infiltration. The findings demonstrate that combinations have better prognostic utility than single chemokines, and may serve as patient stratification tools.

The chemokine CCL5 recruits monocytes into inflamed tissues by triggering primarily CCR1-mediated arrest on endothelial cells, whereas subsequent spreading is dominated by CCR5. The CCL5-induced arrest can be enhanced by heteromer formation with CXCL4. To identify mechanisms for receptor-specific functions, we employed CCL5 mutants and transfectants expressing receptor chimeras carrying transposed extracellular regions. Mutation of the basic 50s cluster of CCL5, a coordinative site for CCL5 surface presentation, reduced CCR5- but not CCR1-mediated arrest and transmigration. Impaired arrest was restored by exchanging the CCR5-N-terminus for that of CCR1, which supported arrest even without the 50s cluster, whereas mutation of the basic 40s cluster essential for proteoglycan binding of CCL5 could not be rescued. The enhancement of CCL5-induced arrest by CXCL4 was mediated by CCR1 requiring its third extracellular loop. The domain exchanges did not affect formation and co-localisation of receptor dimers, indicating a sensing role of the third extracellular loop for hetero-oligomers in an arrest microenvironment. Our data identify confined targetable regions of CCR1 specialised to facilitate CCL5-induced arrest and enhanced responsiveness to the CXCL4-CCL5 heteromer.

OBJECTIVE

IBIS-1 was a pilot study undertaken to correlate coronary imaging with circulating biomarker expression in patients with stable angina, unstable angina and acute myocardial infarction. We hypothesized that patients at high risk of future events could be identified in the future by a combination of high risk plaque features by plaque echogenicity and palpography and a set of circulating blood biomarkers.

RESULTS

We assessed the expression of conventional biomarkers and novel marker protein microarray (170 analytes) over 6 months. There were no strong correlations observed between conventional biomarkers and coronary imaging in non-culprit artery. Proteomic microarray was performed in 66 patients. Seventy eight (45%) analytes showed dynamic changes over time. Using hierarchical clustering and principal component analysis two subsets of biomarkers were identified: initial up-regulation and decrease over time (D-dimer, hepatocyte growth factor, CXCL9/MIG, platelet factor 4/CXCL4, CTACK, C-6 Kine, follistatin, and FGF-7) and the opposite increase (PAI-1- anti-apoptotic protein and I-309--chemokine induced on the human endothelium by Lp(a)).

CONCLUSIONS

Proteomic analysis identifies dynamic patterns in circulating biomarkers in a wide range of patients with coronary artery disease. Further large natural history studies are needed to better define multibiomarker sets for identification of patients at risk of future CV events.

Despite improvements in cancer early detection and treatment, metastatic breast cancer remains deadly. Current therapeutic approaches have very limited efficacy in patients with triple negative breast cancer. Among the many mechanisms associated that contribute to cancer progression, signaling through the CXCL12-CXCR4 is an essential step in cancer cell migration. We previously demonstrated the formation of CXCL12-CXCL4 heterodimers (Carlson et al., 2013). Here, we investigated whether CXCL12-CXCL4 heterodimers alter tumor cell migration. CXCL12 alone dose-dependently promoted the MDA-MB 231 cell migration (p < .05), which could be prevented by blocking the CXCR4 receptor. The addition of CXCL4 inhibited the CXCL12-induced cell migration (p < .05). Using NMR spectroscopy, we identified the CXCL4-CXCL12 binding interface. Moreover, we generated a CXCL4-derived peptide homolog of the binding interface that mimicked the activity of native CXCL4 protein. These results confirm the formation of CXCL12-CXCL4 heterodimers and their inhibitory effects on the migration of breast tumors cells. These findings suggest that specific peptides mimicking heterodimerization of CXCL12 might prevent breast cancer cell migration.

Systemic sclerosis (SSc) is a complex heterogeneous fibrotic autoimmune disease with an unknown exact etiology, and characterized by three hallmarks: fibrosis, vasculopathy, and immune dysfunction. Dendritic cells (DCs) are specialized cells in pathogen sensing with high potency of antigen presentation and capable of releasing mediators to shape the immune response. Altered DCs distributions and their impaired functions may account for their role in breaking the immune tolerance and driving inflammation in SSc, and the direct contribution of DCs in promoting endothelial dysfunction and fibrotic process has only begun to be understood. Plasmacytoid dendritic cells in particular have been implicated due to their high production of type I interferon as well as other cytokines and chemokines, including the pro-inflammatory and anti-angiogenic CXCL4. Furthermore, a deeper understanding of human and mouse DC biology has clarified their identification and function in different tissues, and novel DC subsets have only recently been discovered. In this review, we highlight key findings and recent advances exploring DC role in the pathogenesis of SSc and other related autoimmune diseases, and consideration of their potential use as targeted therapy in SSc.

Infection induces platelet activation and consumption, which leads to thrombocytopenia, enhances microvascular thrombosis, impairs microcirculation and eventually triggers disseminated intravascular coagulation (DIC). It is well characterized that endotoxemia results in a pro-inflammatory and pro-coagulatory state, which favors platelet activation. However the early, direct effects of endotoxemia on platelets have not been investigated so far. Therefore we aimed to determine the early effects of the endotoxin lipopolysaccharide (LPS) on platelet function in vivo. In a human endotoxemia model, 15 healthy volunteers were stimulated with LPS (2 ng/kg). Blood was drawn before, 10, 30 and 60 min after LPS challenge and platelet activation analyzed by flow cytometry (GPIIb/IIIa activation, surface CD62P and CD40L, intraplatelet reactive oxygen formation and platelet-leukocyte aggregates) and ELISA (sCD40L, sCD62P and CXCL4). In parallel, blood samples and platelets were spiked with LPS (50 pg/ml) in vitro and monitored over 60 min for the same platelet activation markers. In vitro platelet stimulation with LPS activated platelets independent of the presence of leukocytes and enhanced their adhesion to endothelial cells. In contrast, in vivo no increase in GPIIb/IIIa activation or surface expression of CD62P was observed. However, endotoxemia resulted in a significant drop in platelet count and elevated the plasma CXCL4 levels already 10 min after the LPS challenge. These data indicate that LPS rapidly activates platelets, leading to α-granule release and endothelial adhesion. This might explain the drop in platelet count observed at the onset of endotoxemia.

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).

RESULTS

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.

CXCL4 chemokines have antiangiogenic properties, mediated by different mechanisms, including CXCR3 receptor activation. Chemokines have distinct oligomerization states that are correlated with their biological functions. CXCL4 exists as a stable tetramer under physiological conditions. It is unclear whether the oligomerization state impacts CXCL4-receptor interaction. We found that the CXCL4 tetramer is sensitive to pH and salt concentration. Residues Glu28 and Lys50 were important for tetramer formation, and the first β-strand and the C-terminal helix are critical for dimerization. By mutating the critical residues responsible for oligomerization, we generated CXCL4 mutants that behave as dimers or monomers under neutral/physiological conditions. The CXCL4 monomer acts as the minimal active unit for interacting CXCR3A, and sulfation of N-terminal tyrosine residues on the receptor is important for binding. Noticeably, CXCL4L1, a CXCL4 variant that differs by three residues in the C-terminal helix, could activate CXCR3A. CXCL4L1 showed a higher tendency to dissociate into monomers, but native CXCL4 did not. This result indicates that monomeric CXCL4 behaves like CXCL4L1. Thus, in this chemokine family, being in the monomeric state seems critical for interaction with CXCR3A.

The pathological events in human cerebral malaria are mimicked in the experimental cerebral malaria (ECM) in Plasmodium berghei ANKA (PBA)-infected C57BL/6 mice. Although previously implied in ECM, the kinetics of cytokines and chemokines expression-an essential functional feature for defining causality in ECM development-remained untested. Herein, we characterized the immunopathological changes and the expression of negative immune regulatory molecules, cytokines and chemokines through asymptomatic (3days after infection, 3dpi), symptomatic (5dpi) and ECM (7dpi) stages in PBA-infected C57BL/6 mice. Parasitized RBCs were first detected in brain on 3dpi, edema and tissue alterations on 5dpi, and hemorrhages in different areas of brain on 7dpi. Increased cerebellar PD-1, CTLA-4 and LAG-3 expression and reduced hippocampal CXCL-4 expression on 3dpi were the first observed immunological changes. The negative immune regulatory molecules (PD-L1, CTLA-4), cytokines (TNF-α, sFAS-L), and chemokines (CXCL-10, MIP-1β) transcript levels varied in different brain areas in symptomatic and ECM phases. By 5dpi, TNF-α, CXCL10 and MIP-1β significantly increased in all brain parts studied; IL-1RA in whole brain, whereas CXCL4 reduced in hippocampus and cerebrum. By 7dpi, the hippocampal PD-1, CXCL4 and CTLA-4 expression decreased but the cerebral, cerebellar and hippocampal PD-L1 expression were elevated. TNF-α, CXCL10, MIP-1β, PD-1, CTLA-4 and PD-L1 expression were up-regulated in different brain areas. The TNFR2, IFN-gamma receptor, Lymphotoxin-β receptor and sFAS-L transcripts significantly increased in brain in ECM. Our data characterize key dynamic immunopathological changes in brain to imply relationship to ECM development.

This study was designed to determine whether irisolidone and its glycoside kakkalide, which are the major constituents of the flower of Pueraria lobata (Kudzu) can attenuate ethanol-induced gastritic injury in mice.

Irisolidone and kakkalide inhibited IL-8 secretion and NF-κB activation in lipopolysaccharide-stimulated KATO III cells. Therefore, we investigated their protective effects against ethanol-induced gastric injury in mice. Pretreatment with kakkalide or irisolidone decreased the area of hemorrhagic ulcerative lesions caused by ethanol and suppressed stomach myeloperoxidase activity, CXCL4 secretion, and NF-κB activation. The ameliorating effect of irisolidone was more potent than that of kakkalide.

Irisolidone may attenuate ethanol-induced gastritis by inhibiting the infiltration of immune cells, particularly neutrophils, through the regulation of CXCL-4 or IL-8 secretion.

Epilepsy is a common neurological disorder with a complex etiology. Our previous study demonstrated that dipeptidyl peptidase IV (DPP4) may be associated with the pathogenesis of epilepsy. However, whether the DPP4 inhibitor sitagliptin has an anticonvulsant effect and the underlying mechanism remain to be elucidated. In this study, we determined that sitagliptin remarkably attenuated the severity of seizures in a pentylenetetrazole (PTZ)-induced rat model. In addition, sitagliptin decreased epileptiform activity measured by electroencephalography (EEG) recordings and patch-clamp methods. Interestingly, sitagliptin pretreatment downregulated the RAGE-JAK2/STAT3 pathway and decreased the expression of CXCL4 and CXCR3. Moreover, CXCR3 knockdown decreased the expression of RAGE, JAK2 and STAT3 in cultured neurons, which suggests that CXCR3 is upstream of the RAGE-JAK2/STAT3 pathway. Altogether, our present data suggest that sitagliptin has an anticonvulsant effect, which might act via downregulation of the CXCL4/CXCR3 axis, followed by a decrease in RAGE and JAK2/STAT3 expression. Considering these effects, sitagliptin could be considered as a novel potential anticonvulsant drug.

DNA Topoisomerase I (TopoI) is a candidate autoantigen for diffuse cutaneous systemic sclerosis (dcSSc) associated with fatal lung disease. Dendritic cells (DCs) contribute to bleomycin-induced lung fibrosis. However, the possibility that TopoI-loaded DCs are involved in the initiation and/or perpetuation of dcSSc has not been explored. Here, we show that immunization with TopoI peptide-loaded DCs induces anti-TopoI autoantibody response and long-term fibrosis. Mice were repeatedly immunized with unpulsed DCs or DCs loaded with either TOPOIA or TOPOIB peptides, selected from different regions of TopoI. At week 12 after initial DC immunization, TOPOIA DCs but not TOPOIB DCs immunization induced mixed inflammation and fibrosis in lungs and skin. At a late time point (week 18), both TOPOIA DCs and TOPOIB DCs groups displayed increased alpha-smooth muscle actin expression in lungs and dermis along with skin fibrosis distal from the site of injection when compared with unpulsed DCs. Both TopoI peptide-DC-immunized groups developed IgG2a anti-TopoI autoantibody response. At week 10, signs of perivascular, peribronchial, and parenchymal pulmonary inflammation were already observed in the TOPOIA DCs group, together with transient elevation in bronchoalveolar lavage cell counts, IL-17A expression, and CXCL4 production, a biomarker of early human dcSSc. Collectively, TopoI peptide DCs induce progressive autoantibody response as well as development of protracted skin and lung dcSSc-like disease. Pronounced lung inflammation, transient IL-17A, and CXCL4 expression precede fibrosis development. Our immunization strategy, that uses self immune system and autoantigen, will help to further investigate the pathogenesis of this complex autoimmune disorder with unmet medical needs.

Human monocytes respond to a variety of stimuli with a complex spectrum of activities ranging from acute defense mechanisms to cell differentiation or cytokine release. However, the individual intracellular signaling pathways related to these functions are not well understood. CXC chemokine ligand 4 (CXCL4) represents a broad activator of monocytes, which induces acute as well as delayed activities in these cells including cell differentiation, survival, or the release of ROS, and cytokines. Here, we report for the first time that CXCL4-treated monocytes significantly upregulate sphingosine kinase 1 (SphK1) mRNA and that CXCL4 induces SphK1 enzyme activity as well as its translocation to the cell membrane. Furthermore, we could show that pharmacological inhibition of SphK results in reversal of CXCL4-induced monocyte survival, cytokine expression, and release of oxygen radicals, which was confirmed by the use of SphK1-specific siRNA. CXCL4-mediated rescue from apoptosis, which is accompanied by inhibition of caspases, is controlled by SphK1 and its downstream element Erk. Taken together, these data assign SphK1 as a central regulator of acute and delayed monocyte activation and suggest SphK1 as a potential therapeutic target to suppress pro-inflammatory responses induced by CXCL4.

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbβ3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbβ3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects.

BACKGROUND

CXCL4 is a platelet chemokine released at micromolar concentrations upon platelet activation. CXCL4 has been shown to promote atherogenesis by various mechanisms. However, data on CXCL4 plasma levels in patients with coronary artery disease are largely inconclusive. Computed coronary artery angiography (CCTA) represents an excellent tool to quantify and characterize coronary atherosclerotic plaques. We hypothesized that increased CXCL4 plasma levels may be associated with features of plaque instability resulting in adverse cardiovascular events. Specifically, we sought to determine whether CXCL4 levels are correlated with specific features of coronary artery disease including (1) plaque volume, (2) calcium score, (3) degree of stenosis, or (4) vascular remodeling.

RESULTS

CXCL4 plasma levels were measured by ELISA in 217 patients undergoing CCTA for suspected CAD (mean age 64.2 ± 9.4 years, 107 (49.3%) male). Mean CXCL4 plasma levels were 12.5 ± 4.6 ng/mL. There was no significant correlation between CXCL4 levels and any clinical or demographic parameters including cardiovascular risk factors. CXCL4 plasma levels did not differ between patient with or without coronary artery disease (CAD: 12.5 ± 4.5 ng/ml, no CAD: 12.5 ± 4.8 ng/ml). Neither univariate nor multivariate analysis showed an association between CXCL4 levels and plaque volume, total calcium score, degree of stenosis, or vascular remodeling. Subgroup analysis of patients with CAD as confirmed by CCTA did not show any association of CXCL4 levels with the extent of CAD.

CONCLUSIONS

While CXCL4 may be present and active within the arterial wall, local increase of CXCL4 may not translate into systemically elevated CXCL4 levels. Further studies will have to test whether CXCL4 may still represent a suitable therapeutic target in human atherosclerosis.

Platelets drive endothelial cell activation in many diseases. However, if this occurs in Plasmodium vivax malaria is unclear. As platelets have been reported to be activated and to play a role in inflammatory response during malaria, we hypothesized that this would correlate with endothelial alterations during acute illness. We performed platelet flow cytometry of PAC-1 and P-selectin. We measured platelet markers (CXCL4, CD40L, P-selectin, Thrombopoietin, IL-11) and endothelial activation markers (ICAM-1, von Willebrand Factor and E-selectin) in plasma with a multiplex-based assay. The values of each mediator were used to generate heatmaps, K-means clustering and Principal Component analysis. In addition, we determined pair-wise Pearson's correlation coefficients to generate correlation networks. Platelet counts were reduced, and mean platelet volume increased in malaria patients. The activation of circulating platelets in flow cytometry did not differ between patients and controls. CD40L levels (Median [IQ]: 517 [406-651] vs. 1029 [732-1267] pg/mL, P = 0.0001) were significantly higher in patients, while P-selectin and CXCL4 showed a nonsignificant trend towards higher levels in patients. The network correlation approach demonstrated the correlation between markers of platelet and endothelial activation, and the heatmaps revealed a distinct pattern of activation in two subsets of P. vivax patients when compared to controls. Although absolute platelet activation was not strong in uncomplicated vivax malaria, markers of platelet activity and production were correlated with higher endothelial cell activation, especially in a specific subset of patients.