Correction: del Molino del Barrio et al. Breast Cancer: An Examination of the Potential of ACKR3 to Modify the Response of CXCR4 to CXCL12. Int. J. Mol. Sci. 2018, 19, 3592.

The authors and Editorial Office were made aware of an error in a figure within the original publication [...].

Dissecting the Therapeutic Mechanisms of Sphingosine-1-Phosphate Receptor Agonism during Ischaemia and Reperfusion.

Sphingosine 1-phosphate (S1P) and S1P receptors (S1PR) regulate many cellular processes, including lymphocyte migration and endothelial barrier function. As neutrophils are major mediators of inflammation, their transendothelial migration may be the target of therapeutic approaches to inflammatory conditions such as ischaemia-reperfusion injury (IRI). The aim of this project was to assess whether these therapeutic effects are mediated by S1P acting on neutrophils directly or indirectly through the endothelial cells. First, our murine model of peritoneum cell recruitment demonstrated the ability of S1P to reduce CXCL8-mediated neutrophil recruitment. Mechanistic in vitro studies revealed that S1P signals in neutrophils mainly through the S1PR1 and 4 receptors and induces phosphorylation of ERK1/2; however, this had no effect on neutrophil transmigration and adhesion. S1P treatment of endothelial cells significantly reduced TNF-α-induced neutrophil adhesion under flow (p < 0.01) and transendothelial migration towards CXCL8 during in vitro chemotaxis assays (p < 0.05). S1PR1 agonist CYM5442 treatment of endothelial cells also reduced neutrophil transmigration (p < 0.01) and endothelial permeability (p < 0.005), as shown using in vitro permeability assays. S1PR3 agonist had no effects on chemotaxis or permeability. In an in vivo mouse model of renal IRI, S1PR agonism with CYM5442 reduced endothelial permeability as shown by reduced Evan's Blue dye extravasation. Western blot was used to assess phosphorylation at different sites on vascular endothelial (VE)-cadherin and showed that CYM5442 reduced VEGF-mediated phosphorylation. Taken together, the results of this study suggest that reductions in neutrophil infiltration during IRI in response to S1P are mediated primarily by S1PR1 signalling on endothelial cells, possibly by altering phosphorylation of VE-cadherin. The results also demonstrate the therapeutic potential of S1PR1 agonist during IRI.

Modeling the Effects of IL-1ß-mediated Inflammation During Ex Vivo Lung Perfusion Using a Split Human Donor Model.

BACKGROUND: The association between interleukin-1ß (IL-1ß) concentrations during ex vivo lung perfusion (EVLP) with donor organ quality and post-lung transplant outcome has been demonstrated in several studies. The mechanism underlying IL-1ß-mediated donor lung injury was investigated using a paired single-lung EVLP model. METHODS: Human lung pairs were dissected into individual lungs and perfused on identical separate EVLP circuits, with one lung from each pair receiving a bolus of IL-1ß. Fluorescently labeled human neutrophils isolated from a healthy volunteer were infused into both circuits and quantified in perfusate at regular timepoints. Perfusates and tissues were subsequently analyzed, with perfusates also used in functional assays. RESULTS: Neutrophil numbers were significantly lower in perfusate samples collected from the IL-1ß-stimulated lungs consistent with increased neutrophil adhesion ( P = 0.042). Stimulated lungs gained significantly more weight than controls ( P = 0.046), which correlated with soluble intercellular adhesion molecule-1 (R 2 = 0.71, P = 0.0043) and von-Willebrand factor (R 2 = 0.39, P = 0.040) in perfusate. RNA expression patterns for inflammatory genes were differentially regulated via IL-1ß. Blockade of IL-1ß significantly reduced neutrophil adhesion in vitro ( P = 0.025). CONCLUSION: These data illustrate the proinflammatory functions of IL-1ß in the context of EVLP, suggesting this pathway may be susceptible to therapeutic modulation before transplantation.

Nitration of chemokine CXCL8 acts as a natural mechanism to limit acute inflammation.

Chemokine CXCL8 is a key facilitator of the human host immune response, mediating neutrophil migration, and activation at the site of infection and injury. The oxidative burst is an important effector mechanism which leads to the generation of reactive nitrogen species (RNS), including peroxynitrite. The current study was performed to determine the potential for nitration to alter the biological properties of CXCL8 and its detection in human disease. Here, we show peroxynitrite nitrates CXCL8 and thereby regulates neutrophil migration and activation. The nitrated chemokine was unable to induce transendothelial neutrophil migration in vitro and failed to promote leukocyte recruitment in vivo. This reduced activity is due to impairment in both G protein-coupled receptor signaling and glycosaminoglycan binding. Using a novel antibody, nitrated CXCL8 was detected in bronchoalveolar lavage samples from patients with pneumonia. These findings were validated by mass spectrometry. Our results provide the first direct evidence of chemokine nitration in human pathophysiology and suggest a natural mechanism that limits acute inflammation.

Understanding Ocular Surface Inflammation in Tears Before and After Autologous Cultivated Limbal Epithelial Stem Cell Transplantation.

INTRODUCTION: We aimed to determine the expression of inflammatory cytokines in the tears of patients with unilateral total limbal stem cell deficiency (TLSCD) caused by chemical burns before and after autologous cultivated limbal epithelial stem cell transplantation (CLET). METHODS: Tear samples were collected from both eyes of 23 patients with unilateral TLSCD and 11 healthy controls, at fixed timepoints before and after CLET. Dissolved molecules were extracted from Schirmer's strips using a standardised method and analysed on an array plate of ten inflammatory cytokines (V-Plex Proinflammatory Panel 1 Human Kit, MSD). RESULTS: IL1ß expression was significantly elevated in the TLSCD eye compared with the unaffected eye at baseline (p < 0.0001) but decreased to normal 3 months post-CLET (p = 0.22). IL6 and IL8 were unaffected at baseline but significantly elevated in the TLSCD eyes at 1 month post-CLET (p = 0.001 and p < 0.0001, respectively). IL6 returned to normal at 3 months and IL8 at 6 months post-CLET. There was a significant renewed increase in IL1ß, IL6 and IL8 expression at 12 months post-CLET (p < 0.0001, p = 0.0001 and p = 0.0003, respectively). IFNγ, IL10 and IL12p70 expression were significantly reduced in both eyes of patients with unilateral TLSCD at all timepoints. CONCLUSION: IL1ß is a specific marker of inflammation in TLSCD eyes that could be therapeutically targeted pre-CLET to improve stem cell engraftment. At 12 months post-CLET the spike in levels of IL1ß, IL6 and IL8 coincides with cessation of topical steroids, suggesting ongoing subclinical inflammation. We therefore recommend not discontinuing topical steroid treatment in cases where penetrating keratoplasty is indicated; however, further investigation is needed to ascertain this. TRIAL REGISTRATION: European Union Drug Regulating Authorities Clinical Trials Database (EuDRACT 2011-000608-16); ISRCTN (International Standard Randomised Controlled Trial Number (isrctn51772481).

MicroRNA antagonist therapy during normothermic machine perfusion of donor kidneys.

Normothermic machine perfusion (NMP) is a novel clinical approach to overcome the limitations of traditional hypothermic organ preservation. NMP can be used to assess and recondition organs prior to transplant and is the subject of clinical trials in solid organ transplantation. In addition, NMP provides an opportunity to deliver therapeutic agents directly to the organ, thus avoiding many limitations associated with systemic treatment of the recipient. We report the delivery of oligonucleotide-based therapy to human kidneys during NMP, in this case to target microRNA function (antagomir). An antagomir targeting mir-24-3p localized to the endothelium and proximal tubular epithelium. Endosomal uptake during NMP conditions facilitated antagomir co-localization with proteins involved in the RNA-induced silencing complex (RISC) and demonstrated engagement of the miRNA target. This pattern of uptake was not seen during cold perfusion. Targeting mir-24-3p action increased expression of genes controlled by this microRNA, including heme oxygenase-1 and sphingosine-1-phosphate receptor 1. The expression of genes not under the control of mir-24-3p was unchanged, indicating specificity of the antagomir effect. In summary, this is the first report of ex vivo gymnotic delivery of oligonucleotide to the human kidney and demonstrates that NMP provides the platform to bind and block detrimental microRNAs in donor kidneys prior to transplantation.

MiR-126-3p Is Dynamically Regulated in Endothelial-to-Mesenchymal Transition during Fibrosis.

In fibrotic diseases, myofibroblasts derive from a range of cell types including endothelial-to-mesenchymal transition (EndMT). Increasing evidence suggests that miRNAs are key regulators in biological processes but their profile is relatively understudied in EndMT. In human umbilical vein endothelial cells (HUVEC), EndMT was induced by treatment with TGFß2 and IL1ß. A significant decrease in endothelial markers such as VE-cadherin, CD31 and an increase in mesenchymal markers such as fibronectin were observed. In parallel, miRNA profiling showed that miR-126-3p was down-regulated in HUVECs undergoing EndMT and over-expression of miR-126-3p prevented EndMT, maintaining CD31 and repressing fibronectin expression. EndMT was investigated using lineage tracing with transgenic Cdh5-Cre-ERT2; Rosa26R-stop-YFP mice in two established models of fibrosis: cardiac ischaemic injury and kidney ureteric occlusion. In both cardiac and kidney fibrosis, lineage tracing showed a significant subpopulation of endothelial-derived cells expressed mesenchymal markers, indicating they had undergone EndMT. In addition, miR-126-3p was restricted to endothelial cells and down-regulated in murine fibrotic kidney and heart tissue. These findings were confirmed in patient kidney biopsies. MiR-126-3p expression is restricted to endothelial cells and is down-regulated during EndMT. Over-expression of miR-126-3p reduces EndMT, therefore, it could be considered for miRNA-based therapeutics in fibrotic organs.

Contribution of Heparan Sulphate Binding in CCL21-Mediated Migration of Breast Cancer Cells.

Chemokine receptor CCR7 is implicated in the metastasis of breast cancer to the lymph nodes. Chemokine function is dependent upon their binding to both cell-surface heparan sulphate (HS) and to their specific receptors; thus, the role of HS in CCR7-mediated lymph node metastasis was investigated by creating a non-HS binding chemokine CCL21 (mut-CCL21). Mut-CCL21 (Δ103-134) induced leukocyte chemotaxis in diffusion gradients but did not stimulate trans-endothelial migration of PBMCs (p < 0.001) and 4T1-Luc cells (p < 0.01). Furthermore, the effect of heparin and HS on the chemotactic properties of wild-type (WT) and mut-CCL21 was examined. Interestingly, heparin and HS completely inhibit the chemotaxis mediated by WT-CCL21 at 250 and 500 µg/mL, whereas minimal effect was seen with mut-CCL21. This difference could potentially be attributed to reduced HS binding, as surface plasmon resonance spectroscopy showed that mut-CCL21 did not significantly bind HS compared to WT-CCL21. A murine model was used to assess the potential of mut-CCL21 to prevent lymph node metastasis in vivo. Mice were injected with 4T1-Luc cells in the mammary fat pad and treated daily for a week with 20 µg mut-CCL21. Mice were imaged weekly with IVIS and sacrificed on day 18. Luciferase expression was significantly reduced in lymph nodes from mice that had been treated with mut-CCL21 compared to the control (p = 0.0148), suggesting the potential to target chemokine binding to HS as a therapeutic option.

Rapid fall in circulating non-classical monocytes in ST elevation myocardial infarction patients correlates with cardiac injury.

Myocardial infarction leads to a rapid innate immune response that is ultimately required for repair of damaged heart tissue. We therefore examined circulating monocyte dynamics immediately after reperfusion of the culprit coronary vessel in STEMI patients to determine whether this correlated with level of cardiac injury. A mouse model of cardiac ischemia/reperfusion injury was subsequently used to establish the degree of monocyte margination to the coronary vasculature that could potentially contribute to the drop in circulating monocytes. We retrospectively analyzed blood samples from 51 STEMI patients to assess the number of non-classical (NC), classical, and intermediate monocytes immediately following primary percutaneous coronary intervention. Classical and intermediate monocytes showed minimal change. On the other hand, circulating numbers of NC monocytes fell by approximately 50% at 90 minutes post-reperfusion. This rapid decrease in NC monocytes was greatest in patients with the largest infarct size (P < .05) and correlated inversely with left ventricular function (r = 0.41, P = .04). The early fall in NC monocytes post-reperfusion was confirmed in a second prospective study of 13 STEMI patients. Furthermore, in a mouse cardiac ischemia model, there was significant monocyte adhesion to coronary vessel endothelium at 2 hours post-reperfusion pointing to a specific and rapid vessel margination response to cardiac injury. In conclusion, rapid depletion of NC monocytes from the circulation in STEMI patients following coronary artery reperfusion correlates with the level of acute cardiac injury and involves rapid margination to the coronary vasculature.

Novel delivery of cellular therapy to reduce ischemia reperfusion injury in kidney transplantation.

Ex vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimize organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC® ) possess potent immunomodulatory properties that could minimize ischemia reperfusion injury. We investigated the potential capability of MAPC cells in kidney NMP. Pairs (5) of human kidneys, from the same donor, were simultaneously perfused for 7 hours. Kidneys were randomly allocated to receive MAPC treatment or control. Serial samples of perfusate, urine, and tissue biopsies were taken for comparison. MAPC-treated kidneys demonstrated improved urine output (P = .009), decreased expression of injury biomarker NGAL (P = .012), improved microvascular perfusion on contrast-enhanced ultrasound (cortex P = .019, medulla P = .001), downregulation of interleukin (IL)-1ß (P = .050), and upregulation of IL-10 (P < .047) and Indolamine-2, 3-dioxygenase (P = .050). A chemotaxis model demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (P < .001). Immunofluorescence revealed prelabeled MAPC cells in the perivascular space of kidneys during NMP. We report the first successful delivery of cellular therapy to a human kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to transplantation.

Ex situ heart perfusion: The past, the present, and the future.

Despite the advancements in medical treatment, mechanical support, and stem cell therapy, heart transplantation remains the most effective treatment for selected patients with advanced heart failure. However, with an increase in heart failure prevalence worldwide, the gap between donor hearts and patients on the transplant waiting list keeps widening. Ex situ machine perfusion has played a key role in augmenting heart transplant activities in recent years by enabling the usage of donation after circulatory death hearts, allowing longer interval between procurement and implantation, and permitting the safe use of some extended-criteria donation after brainstem death hearts. This exciting field is at a hinge point, with 1 commercially available heart perfusion machine, which has been used in hundreds of heart transplantations, and a number of devices being tested in the pre-clinical and Phase 1 clinical trial stage. However, no consensus has been reached over the optimal preservation temperature, perfusate composition, and perfusion parameters. In addition, there is a lack of objective measurement for allograft quality and viability. This review aims to comprehensively summarize the lessons about ex situ heart perfusion as a platform to preserve, assess, and repair donor hearts, which we have learned from the pre-clinical studies and clinical applications, and explore its exciting potential of revolutionizing heart transplantation.

Cell therapy during machine perfusion.

There has been increasing use of organs from extended criteria or donation after circulatory death donors to meet the demands of the transplant waiting list. Over the past decade, there has been considerable progress in technologies to preserve organs prior to transplantation to improve the function of these marginal organs. This has led to the development of normothermic machine perfusion, whereby an organ is perfused with warmed, oxygenated blood and nutrients to resume normal physiological function in an isolated ex-vivo platform. With this advance in preservation comes significant opportunities to recondition, repair and regenerate organs prior to transplantation using cellular therapies. This review aims to discuss the possibilities of machine perfusion technology; highlighting the potential for organ-directed reconditioning and the future avenues for investigation in this field.

Flavin Mononucleotide as a Biomarker of Organ Quality-A Pilot Study.

BACKGROUND: Flavin mononucleotide (FMN), released from damaged mitochondrial complex I during hypothermic liver perfusion, has been shown to be predictive of 90-day graft loss. Normothermic machine perfusion (NMP) and normothermic regional perfusion (NRP) are used for organ reconditioning and quality assessment before transplantation. This pilot study aimed to investigate the changes of FMN levels during normothermic reperfusion of kidneys, livers, and lungs and examine whether FMN could serve as a biomarker to predict posttransplant allograft quality. METHODS: FMN concentrations, in perfusates collected during NMP of kidneys, abdominal NRP, and ex vivo lung perfusion, were measured using fluorescence spectrometry and correlated to the available perfusion parameters and clinical outcomes. RESULTS: Among 7 transplanted kidneys out of the 11 kidneys that underwent NMP, FMN levels at 60 minutes of NMP were significantly higher in the allografts that developed delayed graft function and primary nonfunction (P = 0.02). Fifteen livers from 23 circulatory death donors that underwent NRP were deemed suitable for transplantation. Their FMN levels at 30 minutes of NRP were significantly lower than those not procured for transplantation (P = 0.004). In contrast, little FMN was released during the 8 lung perfusions. CONCLUSIONS: This proof of concept study suggested that FMN in the perfusates of kidney NMP has the potential to predict posttransplant renal function, whereas FMN at 30 minutes of NRP predicts whether a liver would be accepted for transplantation. More work is required to validate the role of FMN as a putative biomarker to facilitate safe and reliable decision-making before embarking on transplantation.

Dual MicroRNA Blockade Increases Expression of Antioxidant Protective Proteins: Implications for Ischemia-Reperfusion Injury.

BACKGROUND: MicroRNAs (miRNAs) are short noncoding RNAs which each cause repression of many target genes. Previous work has demonstrated that therapeutic blockade of single miRNAs is possible. miR-24-3p and miR-145-5p are reported to have a detrimental role in ischemia-reperfusion injury. As the action of miRNAs is inhibitory, we hypothesized that dual blockade of both miRNAs could synergistically upregulate shared target genes. METHODS: Quantification of miRNA expression in donated kidneys was performed using polymerase chain reaction (PCR) panels. Ischemia-reperfusion injury was modeled in vitro by placing human umbilical vein endothelial cells into a hypoxic incubator (1% O2) for 24 hours, with reoxygenation for 6 hours. RNA expression was quantified with reverse transcription quantitative polymerase chain reaction and protein expression assessed with Western blot. Antisense oligonucleotides were used to inhibit miRNAs. RESULTS: miR-24-3p and miR-145-5p were highly expressed in human kidneys following extended cold ischemia. In vitro, hypoxia caused significant upregulation of miR-24-3p (P ≤ 0.001) and miR-145-5p (P ≤ 0.001) and significant downregulation in messenger RNA of shared targets superoxide dismutase 2 (P ≤ 0.001) and heme oxygenase 1 (P ≤ 0.001). These changes were mirrored at the protein level. Dual inhibition of both miR-24-3p and miR-145-5p caused significant upregulation of superoxide dismutase 2 and heme oxygenase 1 protein following hypoxia-reoxygenation; fold change of 3.17 (P ≤ 0.05) and 6.97 (P ≤ 0.05) respectively. Dual inhibition resulted in reduced cellular reactive oxygen species production compared with negative control (P ≤ 0.05) and single blockade of miR-24-3p (P ≤ 0.01) or miR-145-5p (P ≤ 0.05). CONCLUSIONS: Dual blockade of 2 miRNAs can act synergistically to increase the expression of shared gene targets. Dual blockade of miR-24-3p and miR-145-5p represents a novel therapeutic option worthy of further research.

Kinetics Analysis of Circulating MicroRNAs Unveils Markers of Failed Myocardial Reperfusion.

BACKGROUND: Failed myocardial reperfusion occurs in approximately 50% of patients with ST-elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (PPCI). It manifests as microvascular obstruction (MVO) on cardiac magnetic resonance (CMR) imaging. Although prognostically important, MVO is not routinely screened for. Our aim was to investigate the kinetics of circulating short noncoding ribonucleic acids [microRNAs (miRNAs)] following PPCI and their association with MVO in STEMI patients. METHODS: Screening of 2083 miRNAs in plasma from STEMI patients with (n = 6) and without (n = 6) MVO was performed by next-generation sequencing. Two candidate miRNAs were selected and quantified at 13 time points within 3 h postreperfusion in 20 STEMI patients by reverse transcription and quantitative PCR. Subsequently, these 2 miRNAs were measured in a "validation" STEMI cohort (n = 50) that had CMR imaging performed at baseline and 3 months post-PPCI to evaluate their association with MVO. RESULTS: miR-1 and miR-133b were rapidly released following PPCI in a monophasic or biphasic pattern. Both miRNAs were enriched in circulating microparticles. A second miR-1 peak (90-180 min postreperfusion) seemed to be associated with a higher index of microvascular resistance. In addition, miR-1 and miR-133b levels at 90 min post-PPCI were approximately 3-fold (P = 0.001) and 4.4-fold (P = 0.008) higher in patients with MVO, respectively. Finally, miR-1 was significantly increased in a subgroup of patients with worse left ventricular (LV) functional recovery 3 months post-PPCI. CONCLUSIONS: miR-1 and miR-133b levels increase within 3 h of PPCI. They are positively associated with MVO and worse LV functional recovery post-PPCI.

The Role of miR-200b-3p in Modulating TGF-ß1-induced Injury in Human Bronchial Epithelial Cells.

BACKGROUND: Dysregulation of microRNAs (miRNAs) has been implicated in airway diseases where transforming growth factor-ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) may contribute to pathophysiology. Our study investigated the role of miRNA-200b in TGF-ß1-induced EMT in human bronchial epithelial cells. METHODS: NanoString nCounter miRNA assay was used to profile miRNA in control versus TGF-ß1 (1, 4, and 24 h) stimulated BEAS-2B cells. Immortalized primary bronchial epithelial cell line (BEAS-2B cells), human primary bronchial epithelial cells (PBECs), and PBECs derived post-lung transplant were transfected with miR-200b-3p mimics and EMT marker expression was examined at RNA and protein level. miRNA target studies were performed and validated using computational tools and luciferase assay. In situ hybridization was done on normal lung tissue to localize miR-200b-3p in airway epithelium. RESULTS: miR-200b-3p was downregulated post-TGF-ß1 treatment compared with control in BEAS-2B. miR-200b-3p mimic transfection before TGF-ß1 stimulation maintained epithelial marker expression and downregulated mesenchymal cell markers at RNA and protein level in BEAS-2B cells and PBECs. Furthermore, miR-200b-3p mimics reversed established TGF-ß1-induced EMT in BEAS-2B cells. miR-200b-3p targets, ZNF532, and ZEB2 were validated as direct targets using luciferase assay. miR-200b-3p mimics suppress TGF-ß1-induced EMT via inhibition of ZNF532 and ZEB2. In situ hybridization showed that miR-200b-3p is expressed in the normal lung epithelium. Additionally, miR-200b-3p mimics inhibit EMT in the presence of TGF-ß1 in PBECs derived from lung allograft. CONCLUSIONS: We provide proof of concept that miR-200b-3p protects airway epithelial cells from EMT. Manipulating miR-200b-3p expression may represent a novel therapeutic modulator in airway pathophysiology.

A C-terminal CXCL8 peptide based on chemokine-glycosaminoglycan interactions reduces neutrophil adhesion and migration during inflammation.

Leucocyte recruitment is critical during many acute and chronic inflammatory diseases. Chemokines are key mediators of leucocyte recruitment during the inflammatory response, by signalling through specific chemokine G-protein-coupled receptors (GPCRs). In addition, chemokines interact with cell-surface glycosaminoglycans (GAGs) to generate a chemotactic gradient. The chemokine interleukin-8/CXCL8, a prototypical neutrophil chemoattractant, is characterized by a long, highly positively charged GAG-binding C-terminal region, absent in most other chemokines. To examine whether the CXCL8 C-terminal peptide has a modulatory role in GAG binding during neutrophil recruitment, we synthesized the wild-type CXCL8 C-terminal [CXCL8 (54-72)] (Peptide 1), a peptide with a substitution of glutamic acid (E) 70 with lysine (K) (Peptide 2) to increase positive charge; and also, a scrambled sequence peptide (Peptide 3). Surface plasmon resonance showed that Peptide 1, corresponding to the core CXCL8 GAG-binding region, binds to GAG but Peptide 2 binding was detected at lower concentrations. In the absence of cellular GAG, the peptides did not affect CXCL8-induced calcium signalling or neutrophil chemotaxis along a diffusion gradient, suggesting no effect on GPCR binding. All peptides equally inhibited neutrophil adhesion to endothelial cells under physiological flow conditions. Peptide 2, with its greater positive charge and binding to polyanionic GAG, inhibited CXCL8-induced neutrophil transendothelial migration. Our studies suggest that the E70K CXCL8 peptide, may serve as a lead molecule for further development of therapeutic inhibitors of neutrophil-mediated inflammation based on modulation of chemokine-GAG binding.

Regulation of Endothelial-to-Mesenchymal Transition by MicroRNAs in Chronic Allograft Dysfunction.

Fibrosis is a universal finding in chronic allograft dysfunction, and it is characterized by an accumulation of extracellular matrix. The precise source of the myofibroblasts responsible for matrix deposition is not understood, and pharmacological strategies for prevention or treatment of fibrosis remain limited. One source of myofibroblasts in fibrosis is an endothelial-to-mesenchymal transition (EndMT), a process first described in heart development and involving endothelial cells undergoing a phenotypic change to become more like mesenchymal cells. Recently, lineage tracing of endothelial cells in mouse models allowed studies of EndMT in vivo and reported 27% to 35% of myofibroblasts involved in cardiac fibrosis and 16% of isolated fibroblasts in bleomycin-induced pulmonary fibrosis to be of endothelial origin. Over the past decade, mature microRNAs (miRNAs) have increasingly been described as key regulators of biological processes through repression or degradation of targeted mRNA. The stability and abundance of miRNAs in body fluids make them attractive as potential biomarkers, and progress is being made in developing miRNA targeted therapeutics. In this review, we will discuss the evidence of miRNA regulation of EndMT from in vitro and in vivo studies and the potential relevance of this to heart, lung, and kidney allograft dysfunction.

Heparan sulfate in chronic kidney diseases: Exploring the role of 3-O-sulfation.

One of the main feature of chronic kidney disease is the development of renal fibrosis. Heparan Sulfate (HS) is involved in disease development by modifying the function of growth factors and cytokines and creating chemokine gradients. In this context, we aimed to understand the function of HS sulfation in renal fibrosis. Using a mouse model of renal fibrosis, we found that total HS 2-O-sulfation was increased in damaged kidneys, whilst, tubular staining of HS 3-O-sulfation was decreased. The expression of HS modifying enzymes significantly correlated with the development of fibrosis with HS3ST1 demonstrating the strongest correlation. The pro-fibrotic factors TGFß1 and TGFß2/IL1ß significantly downregulated HS3ST1 expression in both renal epithelial cells and renal fibroblasts. To determine the implication of HS3ST1 in growth factor binding and signalling, we generated an in vitro model of renal epithelial cells overexpressing HS3ST1 (HKC8-HS3ST1). Heparin Binding EGF like growth factor (HB-EGF) induced rapid, transient STAT3 phosphorylation in control HKC8 cells. In contrast, a prolonged response was demonstrated in HKC8-HS3ST1 cells. Finally, we showed that both HS 3-O-sulfation and HB-EGF tubular staining were decreased with the development of fibrosis. Taken together, these data suggest that HS 3-O-sulfation is modified in fibrosis and highlight HS3ST1 as an attractive biomarker of fibrosis progression with a potential role in HB-EGF signalling.

Breast Cancer: An Examination of the Potential of ACKR3 to Modify the Response of CXCR4 to CXCL12.

Upon binding with the chemokine CXCL12, the chemokine receptor CXCR4 has been shown to promote breast cancer progression. This process, however, can be affected by the expression of the atypical chemokine receptor ACKR3. Given ACKR3's ability to form heterodimers with CXCR4, we investigated how dual expression of both receptors differed from their lone expression in terms of their signalling pathways. We created single and double CXCR4 and/or ACKR3 Chinese hamster ovary (CHO) cell transfectants. ERK and Akt phosphorylation after CXCL12 stimulation was assessed and correlated with receptor internalization. Functional consequences in cell migration and proliferation were determined through wound healing assays and calcium flux. Initial experiments showed that CXCR4 and ACKR3 were upregulated in primary breast cancer and that CXCR4 and ACKR3 could form heterodimers in transfected CHO cells. This co-expression modified CXCR4's Akt activation after CXCL12's stimulation but not ERK phosphorylation (p < 0.05). To assess this signalling disparity, receptor internalization was assessed and it was observed that ACKR3 was recycled to the surface whilst CXCR4 was degraded (p < 0.01), a process that could be partially inhibited with a proteasome inhibitor (p < 0.01). Internalization was also assessed with the ACKR3 agonist VUF11207, which caused both CXCR4 and ACKR3 to be degraded after internalization (p < 0.05 and p < 0.001), highlighting its potential as a dual targeting drug. Interestingly, we observed that CXCR4 but not ACKR3, activated calcium flux after CXCL12 stimulation (p < 0.05) and its co-expression could increase cellular migration (p < 0.01). These findings suggest that both receptors can signal through ERK and Akt pathways but co-expression can alter their kinetics and internalization pathways.