BACKGROUND

Osteosarcoma (OS) is the most common pediatric bone cancer. Despite advances in treatment regimens, the survival rate remains 60-70%. There is an urgent need to identify prognostic biomarkers, so that targeted therapies can be developed to improve the outcome.

METHODS

Our laboratory has previously identified that circulating serum amyloid A (SAA) and CXC chemokine ligand 4 (CXCL4) are upregulated in patients with OS. In this study, we tested if they could be used as prognostic biomarkers. We used enzyme-linked immunosorbent assays to measure their concentrations in serum samples (n = 233) and immunohistochemistry to examine their expressions in primary tumors (n = 37). Prognostic significance of the serum concentrations and tumor expressions of the biomarkers was then evaluated.

RESULTS

Patients with "high SAA" and "low CXCL4" circulating levels at diagnosis significantly correlated with a worse outcome (HR = 1.68, P = 0.014), which was independent of the metastatic status. These patients also exhibited a significantly higher rate of poor histologic response to chemotherapy. Furthermore, low tumor expression of CXCL4 correlated with poor survival (HR = 3.57, P = 0.005).

CONCLUSIONS

Our results demonstrate that circulating SAA and CXCL4 may serve as prognostic biomarkers in OS. Targeting CXCL4 has been reported, suggesting that it may be exploited as a therapeutic target in OS.

Antimicrobial peptides (AMPs) constitute an important alternative in the search for new treatments against pathogens. We analyzed the sequence variability in cytokine and chemokine proteins to investigate whether these molecules contain a sequence useful in the development of new AMPs. Cluster analysis allowed the identification of tracts, grouped in five categories showing structure and sequence homology. The structure and function relationship among these groups, was analyzed using physicochemical parameters such as length, sequence, charge, hydrophobicity and helicity, which allowed the selection of a candidate that could constitute an AMP. This peptide comprises the C-terminal alpha-helix of chemokines CXCL4/PF-457-70. Far-UV CD spectroscopy showed that this molecule adopts a random conformation in aqueous solution and the addition of 2, 2, 2 trifluoroethanol (TFE) is required to induce a helical secondary structure. The CXCL4/PF-457-70 peptide was found to have antimicrobial activity and very limited hemolytic activity. The mechanism of action was analyzed using model kinetics and molecular dynamics. The kinetic model led to a reasonable assumption about a rate constant and regulatory step on its mechanism of action. Using molecular dynamics simulations, the structural properties the CXCL4/PF-457-70 have been examined in a membrane environment. Our results show that this peptide has a strong preference for binding to the lipid head groups, consequently, increasing the surface density and decreasing the lateral mobility of the lipids alters its functionality.

Ischemic stroke is a disease of aging and causes high mortality or long-term disability. Diminished neurological recovery after stroke in aged subjects is possibly associated with exaggerated non-specific inflammatory reaction. The focus of the present study was on neurobiological and behavioral differences between young and old rats modulated by indomethacin daily treatment starting at four hours after acute cerebral ischemia in animal model. Our results indicate age-independent positive consequences of non-specific inhibition of inflammation by indomethacin including increased NeuN-positive surviving neurons, reduced infarct volume and enhanced neuroprotective response of innate immune system evidenced by increased Iba1 and Anx3 immunoreactivities and moderately activated microglia in the peri-infarcted area. Quite relevantly, the efficacy of therapy with indomethacin was reduced. In the aged rats, specifically indomethacin is ineffective in inhibiting phagocytic activity, which is probably due failure of the aged brain to up-regulate the expression of several cytokines including TNFα and Cxcl4. At protein level, we observed no change of lysosomal ED1 immunoreactivity under treatment. Our study demonstrates the beneficial anti-inflammatory treatment with indomethacin. However, aging blunted the positive effect.

There are multiple causes of liver fibrosis, common ones include ethanol, toxins, and cholestasis. However, whether these different etiologies lead to the same pathological outcomes contain common genetic targets or signaling pathways, the current research has not attracted widespread attention. GSE40041 and GSE55747 were downloaded from the Gene Expression Omnibus (GEO) database. GSE40041 and GSE55747 represent the differential expression profiles in the liver of mice with bile duct ligation (BDL) and carbon tetrachloride (CCl4) induced liver fibrosis models, respectively. By using GEO2R, 701 differential expression genes (DEGs) in GSE40041 and 6540 DEGs in GSE55747 were identified. 260 co-DEGs were shared and extracted for gene ontology (GO) analysis. Through GO analysis, it was found that the regulation of cell migration in biological processes (BPs) was closely related to the pathogenesis of liver fibrosis, and the genes involved in this process include a key gene, chemokine (C-X-C motif) ligand 14 (CXCL14). Subsequently, further bioinformatic analysis showed that CXCL14 may be regulated by miR-122 to participate in the progression of liver fibrosis. Then real-time PCR and western blotting were performed to validate the expression of CXCL14 in liver tissue after liver fibrosis caused by different etiologies (ethanol, CCl4). The expression of CXCL4 in liver fibrosis induced by BDL was verified in another GEO dataset. Basically consistent with our bioinformatics results, our experimental results showed that the expression of CXCL14 was most significantly increased in alcoholic liver fibrosis model, followed by CCl4-induced liver fibrosis, which was also significantly increased in the BDL-induced model. Thus, CXCL14 can act as a common potential genetic target for different liver fibrosis diseases.

Keywords: Bioinformatics; CXCL14; Hepatic stellate cells; Liver fibrosis; miR-122.

Background: Systemic lupus erythematosus (SLE) is a chronic, systemic autoimmune disease that commonly causes kidney damage. Therefore, we measured plasma levels of cytokines that may be related to renal dysfunction in SLE patients.

Methods: To explore the differences between SLE patients with renal dysfunction and healthy volunteers, the levels of cytokines in plasma were screened using a human cytokine antibody array. Then, we chose fourteen of the elevated cytokines for verification with an expanded sample size by a human magnetic Luminex assay. Plasma samples were isolated from SLE patients (n = 72) and healthy volunteers (n = 8).

Results: Cytokine antibody array data showed elevated plasma cytokines in SLE patients with renal dysfunction compared with healthy volunteers. By using the human magnetic Luminex assay, we found that plasma levels of CHI3L1, GDF-15, IGFBP-2, MIF, ST2, TFF3, and uPAR were significantly higher in SLE patients than in healthy volunteers. Plasma levels of CXCL4 were significantly lower in the active group than in the inactive group, and plasma levels of CHI3L1, IGFBP-2, MIF, and MPO were significantly higher in the active group than in the inactive group. We also analyzed the correlation between plasma cytokine levels and the SLEDAI-2K, and our results showed that the plasma levels of the fourteen selected cytokines were weakly correlated or not correlated with the SLEDAI-2K. We further analyzed the correlation between cytokines and renal dysfunction. Plasma levels of GDF-15 and TFF3 were highly positively correlated with serum creatinine levels and 24-hour urine protein levels.

Conclusion: Our data suggest that plasma levels of GDF-15 and TFF3 are potential renal dysfunction markers in SLE patients, but plasma levels of these cytokines are not correlated with the SLEDAI-2K. Further study is warranted to determine how these cytokines regulate inflammatory responses and renal dysfunction in SLE.

Pulsed acoustic cellular expression (PACE) is a treatment that applies focused acoustic shock waves to promote tissue healing. The aim of this study was to assess the effect of PACE treatment on inflammatory responses in a cremaster muscle ischemia/reperfusion injury model. Seventeen cremaster muscle flaps were evaluated in four groups: nonischemic controls (n = 5), 5-hour ischemia controls (n = 4), preischemic (5-hour) PACE conditioning (n = 4), and postischemic (5-hour) PACE conditioning (n = 4). The expression of proinflammatory cytokines (TNFα, IL-6, IL-1α, IL-1β, GM-CSF) and chemokines (CCL3, CCL4, CXCL4) was assessed using TaqMan® real-time PCR. Expression of ELAM-1, VCAM-1, and ICAM-1 was assessed by immunostaining. Preischemic PACE conditioning upregulated expression of IL-6, CCL3, CCL4, and CXCL4, and downregulated expression of TNFα, GM-CSF, and IL-1α. Postischemic PACE conditioning significantly decreased expression of all evaluated genes. Pre- and postischemic PACE conditioning decreased expression of ELAM-1 and ICAM-1. Results of the study indicate that application of PACE conditioning may have a beneficial effect on the recovery of tissues subjected to the ischemia/reperfusion injury. Postischemic PACE conditioning revealed anti-inflammatory effect as confirmed by decreased expression of inflammatory cytokines, chemokines, and cell adhesion molecules (ELAM-1 and ICAM-1) that are responsible for leukocyte recruitment into ischemic tissues. Hence, PACE therapy may be used effectively in clinical practice as a convenient therapeutic strategy to protect tissues against ischemia/reperfusion related injury after microsurgical procedures of free tissue transfers.

RNA sequencing and DNA methylomic profiling were performed after differentiating monocytes for 6 days into moDCs with/without CXCL4 presence. We show that CXCL4 downregulates genes associated with tolerogenicity in DCs including C1Q. Expression profiles of C1Q genes were negatively correlated with their DNA methylation profiles and with immunogenic genes.

Keywords: C1q; CXCL4; DNA methylation; Monocyte-derived dendritic cells; RNA sequencing.

Chemokines and their receptors participate in the development of cancers by enhancing tumor cell proliferation, angiogenesis, invasion, metastasis and penetration of tumor immune cells. It remains unclear whether CXC chemokine ligand 4 (CXCL4)/CXC chemokine receptor 3-B (CXCR3-B) can be used as an independent molecular marker for establishing prognosis for breast cancer patients. We evaluated CXCL4 and CXCR3-B expression in 114 breast cancer tissues and 30 matched noncancerous tissues using immunohistochemistry and western blot, and determined the correlation between their expression and clinicopathologic findings. We observed that breast cancer tissues express CXCL4 strongly and CXCR3-B weakly compared to noncancerous tissues. Strong CXCL4 expression was detected in 94.7% and weak CXCR3-B expression was detected in 78.9% of the tissues. Therefore, CXCL4/CXCR3-B might play a crucial role in breast cancer progression. We found no significant correlation between CXCL4 and age, tumor stage, tumor grade or TNM stage. CXCR3-B was associated significantly with tumor grade. Moreover, the Chi-square test of association showed that the expression of CXCL4/CXCR3-B might be an independent prognostic marker for breast cancer. Therefore, we suggest that CXCR3-B is an indicator of poor prognosis and may also be a chemotherapeutic target.

Chemokines and inflammatory response of endothelial cells is crucial in the development and progression of inflammatory disease. Lipopolysaccharide (LPS) is a well-known factor to trigger inflammatory response and induce damage of endothelial cells. The present study used lipopolysaccharide (LPS)-treated human vascular endothelial cells (HUVECs) to investigate the function of chemokine CXC chemokine ligand 4 (CXCL4) and its receptor CXC chemokine receptor 3 (CXCR3) in inflammatory-induced endothelial injury. LPS exposure (50, 100, 200 ng/ml) to HUVECs induced a dose- and time-dependent increase in CXCL4 and CXCR3 expression at both mRNA and protein levels. The LPS-induced endothelium hyperpermeability was inhibited by the addition of CXCL4 neutralizing antibody. Moreover, the addition of CXCL4 neutralizing antibody abolished the effects of LPS on tight junction (TJ) protein expression (occludin claudin-4 and Zonula occluden-1[ZO-1]) and p38 phosphorylation, which is supported by the observation of increased TJ protein expression and decreased p38 phosphorylation in LPS-treated HUVECs. SB203580, a p38 inhibitor, protected HUVECs from CXCL4-stimulated damage. In conclusion, CXCL4/CXCR3, which was enhanced by LPS, may be involved in endothelial proliferation, apoptosis, and permeability via the p38 signaling pathway.

WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is a genetic autoimmune disorder that results from gain-of-function mutations in the gene encoding chemokine receptor CXCR4. A previous study characterized a patient with WHIM who underwent a chromothriptic event that resulted in spontaneous deletion of the WHIM allele in a single hematopoietic stem cell and subsequent cure of the disease. In this issue of the JCI, Gao et al. extend this work and show that Cxcl4-haplosufficient bone marrow has a selective advantage for long-term engraftment in murine WHIM models. Moreover, successful engraftment occurred without prior conditioning of recipients. Together, these results have important implications for improving hematopoietic stem/progenitor cell transplant not only for patients with WHIM but also for all patients who may require the procedure.

UNASSIGNED

To study the expression of angiodrastic chemokines in colorectal tumors and correlate findings with clinicopathological parameters and survival.

UNASSIGNED

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.

UNASSIGNED

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.

UNASSIGNED

(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.

Repair of the endometrial surface at menstruation must be efficient to minimize blood loss and optimize reproductive function. The mechanism and regulation of endometrial repair remain undefined.

To determine the presence/regulation of CXCL4 in the human endometrium as a putative repair factor at menses.

Endometrial tissue was collected throughout the menstrual cycle from healthy women attending the gynecology department. Menstrual blood loss was objectively measured in a subset, and heavy menstrual bleeding (HMB) was defined as >80 mL per cycle. Monocytes were isolated from peripheral blood.

CXCL4 messenger RNA (mRNA) and protein were identified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The function/regulation of endometrial CXCL4 was explored by in vitro cell culture.

CXCL4 mRNA concentrations were significantly increased during menstruation. Intense staining for CXCL4 was detected in late secretory and menstrual tissue, localized to stromal, epithelial and endothelial cells. Colocalization identified positive staining in CD68+ macrophages. Treatment of human endometrial stromal and endothelial cells (hESCs and HEECs, respectively) with steroids revealed differential regulation of CXCL4. Progesterone withdrawal resulted in significant increases in CXCL4 mRNA and protein in hESCs, whereas cortisol significantly increased CXCL4 in HEECs. In women with HMB, CXCL4 was reduced in endothelial cells during the menstrual phase compared with women with normal menstrual bleeding. Cortisol-exposed macrophages displayed increased chemotaxis toward CXCL4 compared with macrophages incubated with estrogen or progesterone.

These data implicate CXCL4 in endometrial repair after menses. Reduced cortisol at the time of menses may contribute to delayed endometrial repair and HMB, in part by mechanisms involving aberrant expression of CXCL4.

Some CXC chemokines, including CXCL14, transport CpG oligodeoxynucleotides (ODNs) into dendritic cells (DCs), thereby activating TLR9. The molecular basis of this noncanonical function of CXC chemokines is not well understood. In this study, we investigated the CpG ODN binding and intracellular transport activities of various CXC chemokines and partial peptides of CXCL14 in mouse bone marrow-derived dendritic cells. CXCL14, CXCL4, and CXCL12 specifically bound CpG ODN, but CXCL12 failed to transport it into cells at low dose. CXCL14 N-terminal peptides 1-47, but not 1-40, was capable of transporting CpG ODN into the cell, resulting in an increase in cytokine production. However, both the 1-47 and 1-40 peptides bound CpG ODN. By contrast, CXCL14 peptides 13-50 did not possess CpG ODN binding capacity or transport activity. The chimeric peptides CXCL12 (1-22)-CXCL14 (13-47) bound CpG ODN but failed to transport it. These results suggest that amino acids 1-12 and 41-47 of CXCL14 are required for binding and intracellular transport of CpG ODN, respectively. We found that an anti-CXCL14 Ab blocked cell-surface binding and internalization of the CpG ODN/CXCL14 complex. On the basis of these findings, we propose that CXCL14 has two functional domains, one involved in DNA recognition and the other in internalization of CXCL14-CpG DNA complex via an unidentified CXCL14 receptor, which together are responsible for eliciting the CXCL14/CpG ODN-mediated TLR9 activation. These domains could play roles in CXCL14-related diseases such as arthritis, obesity-induced diabetes, and various types of carcinoma.

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.

Keywords: CXCL4-L1; IFN-I signature; Systemic Sclerosis; Very Early Diagnosis of Systemic Sclerosis (VEDOSS); autoantibodies; autoimmune diseases; biomarkers; chemokine (C-X-C motif) ligand 4 (CXCL4).

BACKGROUND

Poncirin (PO) and isosakuranetin (or ponciretin [PT]) are compounds found in fruits of the genus Citrus. They are frequently used in traditional Chinese medicine for the treatment of inflammation and asthma. Therefore, we examined their anti-gastritis effects in vitro and in vivo.

METHODS

The anti-inflammatory effects of PO and PT were examined using ethanol- or LPS-stimulated KATO III cells. Gastritis was induced in ICR mice via intragastric injection of absolute ethanol. Levels of inflammatory markers were measured by enzyme-linked immunosorbent assay, immunoblotting, and quantitative polymerase chain reaction.

RESULTS

Treatment with PT or PO inhibited the secretion of interleukin (IL)-8 and tumor necrosis factor (TNF) in ethanol- or LPS-stimulated KATO III cells. They also reduced the activation of nuclear factor kappa B (NF-κB). Pre-treatment with PT or PO significantly protected against ethanol-induced hemorrhagic gastritis, characterized by edema, tissue erosions, and mucosal friability in mice. Treatment with PT or PO suppressed ethanol-induced NF-κB activation and the release of TNF, IL-8, and IFN-γ. The protective effect of PT was greater than that of PO and comparable to ranitidine, a positive control.

CONCLUSIONS

PT may attenuate ethanol-induced gastritis by inhibiting the infiltration of immune cells, including neutrophils, via the regulation of CXCL4 (or IL-8) secretion and the activation NF-κB.

Platelets contribute to the regulation of tissue neovascularization, although the specific factors underlying this function are unknown. Here, we identified the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) on platelets as a negative regulatory mechanism of vessel formation. We showed that platelets expressing C5aR1 exert an inhibitory effect on endothelial cell functions such as migration and 2D and 3D tube formation. Growth factor- and hypoxia-driven vascularization was markedly increased in C5ar1^-/- mice. Platelet-specific deletion of C5aR1 resulted in a proangiogenic phenotype with increased collateralization, capillarization and improved pericyte coverage. Mechanistically, we found that C5a induced preferential release of CXC chemokine ligand 4 (CXCL4, PF4) from platelets as an important antiangiogenic paracrine effector molecule. Interfering with the C5aR1-CXCL4 axis reversed the antiangiogenic effect of platelets both in vitro and in vivo.In conclusion, we identified a mechanism for the control of tissue neovascularization through C5a/C5aR1 axis activation in platelets and subsequent induction of the antiangiogenic factor CXCL4.

The cause of atherothrombosis is rupture or erosion of atherosclerotic lesions, leading to an increased risk of myocardial infarction or stroke. Here, platelet activation plays a major role, leading to the release of bioactive molecules, for example, chemokines and coagulation factors, and to platelet clot formation. Several antiplatelet therapies have been developed for secondary prevention of cardiovascular events, in which anticoagulant drugs are often combined. Besides playing a role in hemostasis, platelets are also involved in inflammation. However, it is unclear whether current antiplatelet therapies also affect platelet immune functions. In this study, the possible anti-inflammatory effects of antiplatelet medications on chemokine release were investigated using enzyme-linked immunosorbent assay and on the chemotaxis of THP-1 cells toward platelet releasates. We found that antiplatelet medication acetylsalicylic acid (ASA) led to reduced chemokine (CC motif) ligand 5 (CCL5) and chemokine (CXC motif) ligand 4 (CXCL4) release from platelets, while leukocyte chemotaxis was not affected. Depending on the agonist, α _IIb β ₃ and P2Y ₁₂ inhibitors also affected CCL5 or CXCL4 release. The combination of ASA with a P2Y ₁₂ inhibitor or a phosphodiesterase (PDE) inhibitor did not lead to an additive reduction in CCL5 or CXCL4 release. Interestingly, these combinations did reduce leukocyte chemotaxis. This study provides evidence that combined therapy of ASA and a P2Y ₁₂ or PDE3 inhibitor can decrease the inflammatory leukocyte recruiting potential of the releasate of activated platelets.

Keywords: antiplatelet agents; atherosclerosis; chemokines; inflammation; monocytes; platelets.

Galectins are versatile glycan-binding proteins involved in immunomodulation. Evidence suggests that galectins can control the immunoregulatory function of cytokines and chemokines through direct binding. Here, we report on an inverse mechanism in which chemokines control the immunomodulatory functions of galectins. We show the existence of several specific galectin-chemokine binding pairs, including galectin-1/CXCL4. NMR analyses show that CXCL4 binding induces changes in the galectin-1 carbohydrate binding site. Consequently, CXCL4 alters the glycan-binding affinity and specificity of galectin-1. Regarding immunomodulation, CXCL4 significantly increases the apoptotic activity of galectin-1 on activated CD8⁺ T cells, while no effect is observed in CD4⁺ T cells. The opposite is found for another galectin-chemokine pair, i.e., galectin-9/CCL5. This heterodimer significantly reduces the galectin-9 induced apoptosis of CD4⁺ T cells and not of CD8⁺ T cells. Collectively, the current study describes an immunomodulatory mechanism in which specific galectin-chemokine interactions control the glycan-binding activity and immunoregulatory function of galectins.

Background: Inflammation is the key pathophysiological mechanism of the initiation and progression of atherosclerosis. The study objective was to assess the effects of a dietary intervention based on the model of the dietary approaches to stop hypertension (DASH) diet on the levels of chemokines RANTES and CXCL4 in patients with non-obstructive coronary artery disease.

Methods: As part of Dietary Intervention to Stop Coronary Atherosclerosis in Computed Tomography (DISCO-CT) study, patients were randomised to an intervention group (n = 40), where the DASH diet was introduced along with optimal pharmacotherapy, and to a control group (n = 39), with optimal pharmacotherapy alone. In the DASH group, systematic dietary counselling was provided for the follow-up period. RANTES and CXCL4 levels were determined using ELISA.

Results: In the DASH group, the RANTES level insignificantly reduced from 42.70 ± 21.1 ng/mL to 38.09 ± 18.5 ng/mL (p = 0.134), and the CXCL4 concentration significantly reduced from 12.38 ± 4.1 ng/mL to 8.36 ± 2.3 ng/mL (p = 0.0001). At the same time, an increase in the level of both chemokines was observed in the control group: RANTES from 34.69 ± 22.7 to 40.94 ± 20.0 ng/mL (p = 0.06) and CXCL4 from 10.98 ± 3.6 to 13.0 5± 4.8 ng/mL (p = 0.009). The difference between the changes in both groups was significant for both RANTES (p = 0.03) and CXCL4 (p = 0.00001). The RANTES/CXCL4 ratio reduced in the control group (from 3.52 ± 2.8 to 3.35 ± 2.8; p = 0.006), while in the DASH group, an increase was observed (from 3.54 ± 1.7 to 4.77 ± 2.4; p = 0.001).

Conclusions: A 12-month-long intensive dietary intervention based on DASH diet guidelines as an addition to optimal pharmacotherapy causes changes in the levels of chemokines CXCL4 and RANTES and their mutual relationship in comparison to conventional treatment.

Keywords: DASH diet; atherosclerosis; chemokine CXCL4; chemokine RANTES; inflammation.

Context: Primary hypercholesterolemia (PH) is a lipid disorder characterized by elevated levels of cholesterol and low-density lipoprotein (LDL). Low-grade systemic inflammation is associated with PH, which might explain the higher incidence of cardiovascular diseases in this setting. Objective: To evaluate the effect of an oral unsaturated fat load (OUFL) on different immune parameters and functional consequences in patients with PH in postprandial state. Design: A commercial liquid preparation of long-chain triglycerides (Supracal^®; ωωResults: The PH group had a lower percentage of activated platelets and circulating type 1 monocytes, and blunted neutrophil activation after the OUFL, accompanied by a significant increase in the percentage of regulatory T lymphocytes. In this group, the OUFL led to a significant impairment of leukocyte adhesion to the dysfunctional [tumor necrosis factor α (TNFα)-stimulated] endothelium and reduced the plasma levels of soluble P-selectin, platelet factor-4 (PF-4)/CXCL4, CXCL8, CCL2, CCL5, and TNFα. Conclusion: The OUFL has a beneficial impact on the pro-thrombotic and pro-inflammatory state of PH patients and might be a promising macronutrient approach to dampen the systemic inflammation associated with PH and the development of further cardiovascular events.

Keywords: endothelial dysfunction; inflammatory mediators; leukocytes; oral unsaturated fat load; platelet activation; primary hypercholesterolemia; systemic inflammation.

The chemokines CCL5 and CXCL4 are deposited by platelets onto endothelial cells, inducing monocyte arrest. Here, the fate of CCL5 and CXCL4 after endothelial deposition was investigated. Human umbilical vein endothelial cells (HUVECs) and EA.hy926 cells were incubated with CCL5 or CXCL4 for up to 120 min, and chemokine uptake was analyzed by microscopy and by ELISA. Intracellular calcium signaling was visualized upon chemokine treatment, and monocyte arrest was evaluated under laminar flow. Whereas CXCL4 remained partly on the cell surface, all of the CCL5 was internalized into endothelial cells. Endocytosis of CCL5 and CXCL4 was shown as a rapid and active process that primarily depended on dynamin, clathrin, and G protein-coupled receptors (GPCRs), but not on surface proteoglycans. Intracellular calcium signals were increased after chemokine treatment. Confocal microscopy and ELISA measurements in cell organelle fractions indicated that both chemokines accumulated in the nucleus. Internalization did not affect leukocyte arrest, as pretreatment of chemokines and subsequent washing did not alter monocyte adhesion to endothelial cells. Endothelial cells rapidly and actively internalize CCL5 and CXCL4 by clathrin and dynamin-dependent endocytosis, where the chemokines appear to be directed to the nucleus. These findings expand our knowledge of how chemokines attract leukocytes to sites of inflammation.

Keywords: RANTES; chemokine; endothelial cell; monocyte; platelet factor 4.

The chemokine CXCL4/platelet factor 4 (PF4) gene, a key player in myelofibrosis, was knocked out by CRISPR/Cas9 in induced pluripotent stem cells (iPS cells) of a polycythemia vera (PV) patient with JAK2 V617F mutation. Two CXCL4^KO iPS cell lines with and without JAK2 V617F mutation (UKAi002-B-1 and UKAi002-A-1, respectively) were generated. CXCL4^KO iPS cells showed deletion of exon 1 and complete loss of CXCL4 protein. Pluripotency of iPS cells was confirmed by expression of pluripotency markers and trilineage differentiation. CXCL4^KO iPS cells are expected to provide a valuable tool for investigating the role of CXCL4 in human diseases.

Keywords: CXCL4; Hematopoiesis; Induced pluripotent stem cell; JAK2 V617F; PF4; iPS cells.