CXCR4 mRNA Research Articles

Increased CXCL12 expression in endometrium of women with abnormal uterine bleeding is post-transcriptionally mediated via miR-23b-3p and is associated with decreased expression of the miR-23b-3p/24-3p/27b-3p cluster: a pilot study

To study C-X-C motif chemokine 12 (CXCL12) and CXCR4 expression in endometrial tissue from both women with and without abnormal uterine bleeding (AUB) of endometrial origin and evaluate their relationship with microRNA (miRNA). Retrospective and laboratory study. University-based research laboratory. Nine women with and without abnormal uterine bleeding, all of whom were in the secretory stage of their menstrual cycle, who provided endometrial biopsy tissue. Immunohistochemical localization of CXCL12 and CXCR4 as well as quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assessment of mRNA expression in archived endometrial biopsy tissue and invitro cell culture using the immortalized endometrial stromal cell line, t-HESC. Endometrial stromal cell line, t-HESC transfection with nontargeting, negative control miRNA mimics or miRNA mimics for miR-23b-3p and mRNA assessment miR-23b-3p expression confirmed by qRT-PCR and evaluation of impact on CXCL12 expression at the protein level by enzyme-linked immunosorbent assay and mRNA levels by qRT-PCR. Expression of CXCL12 and CXCR4 protein via immunohistochemistry and mRNA and miRNA levels of CXCL12 and CXCR4 as well as miR-23b-3p, miR-24b-3p, and miR-27b-3p, respectively, via qRT-PCR. CXCL12 and its receptor CXCR4 expression were up-regulated in the endometrial tissue of women with AUB at the protein level, but this up-regulation of expression was only associated with increased CXCR4 mRNA expression. To evaluate whether CXCL12 may be post-transcriptionally regulated, we assessed expression of miR-23b-3p, a bona fide post-transcriptional regulator of CXCL12 expression. The expression of miR-23b-3p was statistically significantly lower in AUB endometrial tissue, as were fellow cluster members miR-24-3p and miR-27-3p. Transfection of t-HESC cells with pre-miR-23b-3p mimics statistically significantly reduced the levels of CXCL12 secreted protein but not mRNA levels, suggesting that miR-23b-3p retards protein translation independent of transcript degradation. Reduced expression of the miR-23b-3p/24-3p/27b-3p cluster is associated with elevated expression of CXCL12, which may contribute to the pathophysiology of AUB.

A Potential Role for CXCR2 in Early-onset Preeclampsia: Placental CXCR2 Expression is Related to Increased Blood Pressure and Serum LDH Levels

Abstract Objective: This study was aimed to determine the changes in CXCR2 expression in preeclampsia placenta and its correlation with clinical parameters. Methods: Sixty-four gravidas ranging in age from 25 to 42 years referred to the obstetrics unit of the West China Second University Hospital from April 2012 to October 2012 were recruited in this case-control study; women were diagnosed and divided into early-onset preeclampsia group (n = 22), late-onset preeclampsia group (n = 22), and healthy pregnancy group (n = 20). After immunolocalized in human placenta, the levels of CXCR2 protein and messenger ribonucleic acid (mRNA) were detected by enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction. Correlations between placental CXCR2 protein expression with systolic blood pressure and lactate dehydrogenase (LDH) in early-onset preeclampsia were examined using Pearson or Spearman's correlation coefficients. Results: Placental CXCR2 protein and mRNA expression in early-onset preeclampsia was significantly lower than it was in placentas from healthy pregnancy pregnancies and late-onset preeclampsia (P < 0.05). The placental CXCR2 protein expression correlated negatively with systolic blood pressure and LDH in early-onset preeclampsia (r = −0.51, P < 0.05; r = −0.43, P < 0.05). Conclusion: Significant abnormal placental CXCR2 expression in early-onset preeclampsia, and its correlations with some clinical parameters (systolic blood pressure and LDH) were discovered, suggesting that CXCR2 may play a role in the pathogenesis of early-onset preeclampsia.

Effect of CXCR5-Positive Cell Infiltration on the Immune Contexture and Patient Prognosis in Head and Neck Squamous Cell Carcinoma.

PurposeCXCR5-positive (CXCR5+) tumor cell infiltration has different prognostic values in different types of cancer. The objective was to evaluate the effect of CXCR5+ cell infiltration in head and neck squamous cell carcinoma (HNSCC).Patients and MethodsThe study included two patient cohorts: The Cancer Genome Atlas cohort (TCGA, n = 472) and the Renji Hospital cohort (RJHC, n = 201). The TCGA and RJHC cohorts were analyzed for CXCR5-related mRNAs and CXCR5+ cell infiltration, respectively. We then evaluated the correlation between CXCR5 mRNA and CXCR5+ cell infiltration in terms of overall survival and the immune contexture.ResultsThe 5-year overall survival rate was significantly correlated with high CXCR5 mRNA expression and CXCR5+ cell infiltration in the TCGA and RJHC cohorts, respectively (p < 0.01), even after adjusting for confounders. Moreover, high CXCR5 mRNA expression was associated with more CD4+ T cells, CD8+ T cells, plasma cells, and less dendritic cells. A high CXCR5 mRNA expression was also correlated with increased expression of cytotoxic IFNG, TNFSF11 (RANKL), GZMA, GZMB, GZMK, GZMM, and PRF1 and increased expression of the immunosuppressive gene PDCD1 (PD-1), CD274 (PD-L1), CTLA4, LAG3, HAVCR2 (TIM-3), BTLA, and TIGIT.ConclusionHNSCC patients with a high intratumoral CXCR5 expression had a better prognosis than those with low intratumoral CXCR5 expression. Moreover, CXCR5+ cell infiltration could be used as an independent prognostic biomarker or as a potential therapeutic target. The presence of CXCR5+ cells affects the infiltration of immunocytes in head and neck cancer, differently from what was reported in other cancer types. Further randomized controlled trials or studies with more patients are needed to validate our results.

CXCR4-overexpressing Mesenchymal Stem Cells exhibit increased cell migration and cytokine secretion

Background & Aim The regenerative and immunomodulatory properties of Mesenchymal Stem Cells (MSCs) have been demonstrated extensively in vitro, and in vivo in various types of pre-clinical models. However, in clinical settings, while the safety profile of MSCs has been well-established, the efficacy data obtained in pre-clinical studies hasn't been recapitulated in most cases where MSCs have been administered to human subjects. Many factors are thought to hinder MSC-based therapies; low potency and retention, and limited homing to the site of injury. In the current study, we sought to engineer bone-marrow-derived MSCs (BMSCs) with improved migratory properties so as to facilitate homing to the site of injury. Methods, Results & Conclusion We utilized a genetic approach in which BMSCs were transfected with CXCR4 mRNA. CXCR4, a G-protein coupled receptor highly expressed on the surface of multiple cell types is best known for its role in hematopoiesis and leukocyte chemotaxis. We showed that transfection of CXCR4 mRNA into BMSCs for eight hours resulted in over 60% of cells positive for CXCR4 protein expression, as determined by flow cytometry. Less than 1% of un-transfected BMSCs expresses CXCR4 on their surface. The expression of CXCR4 was maintained post-thaw when transfected BMSCs were recovered from LN2 Storage. Transfected cells also maintained their viability post-thaw. In terms of functionality, we demonstrated that BMSCs expressing CXCR4 were more migratory compared to their naive counterparts lacking CXCR4. This effect on migration was augmented when CXCR4-BMSCs were induced to migrate towards SDF-1α, the cognate ligand for CXCR4. In addition to the effects on directed cell migration, with the use of a membrane-based antibody array, we showed that compared to control BMSCs, CXCR4-BMSCs secrete greater quantities of MCP-1, a key chemokine that regulates macrophage migration and infiltration. In summary, we have shown that transfection of BMSCs with CXCR4 mRNA increases directed cell migration and alters cytokine secretion. This is a potential strategy for generating BMSCs with superior homing properties for use in clinical settings.

Expression and regulation effects of chemokine receptor 7 in colon cancer cells.

In China the incidence and mortality rates of colon cancer have been increasing annually. Studies have revealed that CXCR7 is expressed in many tumors. The aim of the present study was to investigate the function of CXCR7 in colon cancer. The expression level of chemokine receptor 7 (CXCR7) in Caco-2 and HCT116 cells was investigated to elucidate the effect of CXCR7 on cell biological behavior. Reverse transcription-quantitative PCR and western blot analysis were used to detect the expression level of CXCR7 in Caco-2 and HCT116 cells after transfection with small interfering (si)RNA. To analyze the in vitro biological function of CXCR7, cell proliferation was measured using a Cell Counting Kit-8 assay, and cell invasion and migration were measured using Matrigel, and Transwell and wound healing assays. siRNAs were successfully transfected into Caco-2 and HCT116 cells and resulted in a decrease in CXCR7 protein and mRNA expression. Downregulation of CXCR7 inhibited Caco-2 and HCT116 cell proliferation, invasion, and migration. Regulation of CXCR7 expression may affect the biological behavior of Caco-2 and HCT116 cells, suggesting that CXCR7 has a potential role in molecular therapy in colon cancer.

Selective Upregulation of Transcripts for Six Molecules Related to T Cell Costimulation and Phagocyte Recruitment and Activation among 734 Immunity-Related Genes in the Brain during Perforin-Dependent, CD8+ T Cell-Mediated Elimination of Toxoplasma gondii Cysts.

We recently found that an invasion of CD8+ cytotoxic T cells into tissue cysts of Toxoplasma gondii initiates an elimination of the cysts in association with an accumulation of microglia and macrophages. In the present study, we compared mRNA levels for 734 immune-related genes in the brains of infected SCID mice that received perforin-sufficient or -deficient CD8+ immune T cells at 3 weeks after infection. At 7 days after the T cell transfer, mRNA levels for only six genes were identified to be greater in the recipients of the perforin-sufficient T cells than in the recipients of the perforin-deficient T cells. These six molecules included two T cell costimulatory molecules, inducible T cell costimulator receptor (ICOS) and its ligand (ICOSL); two chemokine receptors, C-X-C motif chemokine receptor 3 (CXCR3) and CXCR6; and two molecules related to an activation of microglia and macrophages, interleukin 18 receptor 1 (IL-18R1) and chitinase-like 3 (Chil3). Consistently, a marked reduction of cyst numbers and upregulation of ICOS, CXCR3, CXCR6, IL-18R1, and Chil3 mRNA levels were also detected when the perforin-sufficient CD8+ immune T cells were transferred to infected SCID mice at 6 weeks after infection, indicating that the CD8+ T cell-mediated protective immunity is capable of eliminating mature T. gondii cysts. These results together suggest that ICOS-ICOSL interactions are crucial for activating CD8+ cytotoxic immune T cells to initiate the destruction of T. gondii cysts and that CXCR3, CXCR6, and IL-18R are involved in recruitment and activation of microglia and macrophages to the T cell-attacked cysts for their elimination.IMPORTANCE T. gondii establishes a chronic infection by forming tissue cysts, which can grow into sizes greater than 50 μm in diameter as a consequence of containing hundreds to thousands of organisms surrounded by the cyst wall within infected cells. Our recent studies using murine models uncovered that CD8+ cytotoxic T cells penetrate into the cysts in a perforin-dependent manner and induce their elimination, which is accompanied with an accumulation of phagocytic cells to the T cell-attacked target. This is the first evidence of the ability of the T cells to invade into a large target for its elimination. However, the mechanisms involved in anticyst immunity remain unclear. Immune profiling analyses of 734 immune-related genes in the present study provided a valuable foundation to initiate elucidating detailed molecular mechanisms of the novel effector function of the immune system operated by perforin-mediated invasion of CD8+ T cells into large targets for their elimination.

TNF Receptor: Fc Fusion Protein Downregulates RANKL/OPG Ratio by Inhibiting CXCL16/CXCR6 in Active Ankylosing Spondylitis.

Clinical studies indicate that recombinant tumor necrosis factor receptor:Fc fusion protein (rhTNFR:Fc) quickly alleviates symptoms and physical signs of active Ankylosing Spondylitis (AS), improving the manifestation of spinal inflammation on radiological imaging. However, the regulatory mechanism of rhTNFR:Fc in the chemokine pathway is unclear. Thus we study the mechanism of phlogogenic activity of CXCL16/CXCR6 in AS and the related mechanism of rhTNFR: Fc treatment. Thirty-two cases of active AS were treated with rhTNFR:Fc for 3 consecutive months. Clinical response was evaluated at baseline and after treatment. CXCL16/CXCR6 expression as well as Receptor Activator Of Nuclear Factor-Κb Ligand (RANKL)/Osteoprotegerin (OPG), essential molecules for osteoclast differentiation, were studied in AS before and after treatment. Further, the proliferation of lymphocytes and the RANKL level stimulated by recombinant human CXCL16 (rhCXCL16) were measured in vitro. Thirty cases responded to rhTNFR:Fc treatment. The RANKL level, RANKL/OPG ratio, CXCLl6 level in serum, and CXCLl6 and CXCR6 mRNA levels in active AS were higher than those in controls and treated patients (P<0.001). rhCXCL16 treatment increased lymphocyte proliferation and RANKL level in active AS (P<0.001), but not in controls or treated patients (P>0.05). A positive linear correlation was noted between CXCL16 serum levels and RANKL/OPG ratio and between CXCL16 levels and C-reactive protein results (P<0.001). Our findings suggest that rhTNFR:Fc suppresses inflammation and bone destruction of AS by reducing the RANKL/OPG ratio through inhibition of the CXCL16/CXCR6 pathway.

Mechanisms underlying the improvement of preeclampsia through salvianolic acid B-regulated miRNA-155/CXCR4.

The aim of this study was to evaluate the effects and mechanisms of salvianolic acid B (Sal B) in preeclampsia treatment by in vivo and in vitro study. Rats were randomly divided into 5 groups. In order to establish the model of preeclampsia, endotoxin was administered to the rats in the Sal B intervention and model groups. The systolic blood pressure (SBP) of the tail artery and urine protein concentration were observed at different points, the miRNA-155 and CXCR4 gene expression levels by RT-PCR and the CXCR4 and p-AKT protein expression by WB assay. Using HTR8/SVneo to explain the mechanisms; evaluating the miRNA-155 and CXCR4 mRNA expression by RT-PCR assay, measuring the cell invasion and migration by transwell and wound healing assay in different groups; evaluating the CXCR4 and p-AKT protein expression by WB assay and p-AKT nucleation volume by cellular immunofluorescence were evaluated. Compared with the normal group, the systolic blood pressure and urine protein were significantly increased in the model group (p < 0.05), serum NO concentration was significantly down-regulated (all p < 0.05), CXCR4 and miRNA-155 mRNA expression was significantly different and CXCR4 and p-AKT protein expression was significantly suppressed (all p < 0.05). With Sal B supplement, the SBP, urine protein and NO concentration were significantly improved with dose-dependent (all p < 0.05). In the cell experiment, the cell invasion and migration ability were significantly improved with Sal B supplement (both p < 0.05). However, with miRNA-155 transfection, the cell invasion and migration ability were suppressed with Sal B treatment (both p < 0.05). Sal B improved preeclampsia via regulation of miRNA-155/CXCR4 in the in vitro and vivo study.

Imipramine and Venlafaxine Differentially Affect Primary Glial Cultures of Prenatally Stressed Rats.

Here, we examine the effects of prenatal administration of two antidepressants—imipramine (IMI) and venlafaxine (VEN)—on morphology and activity of a primary glial culture. Microglia are targeted by antidepressants used for antenatal depression and are important regulators of central nervous system development. In this study, female Wistar rats were assigned to one of four groups: a control group that received water ad libitum (1), and groups that received additionally once daily either water (2), IMI (10 mg/kg) (3), or VEN (20 mg/kg) (4) by oral gavage from gestation day 7 to 22. Oral gavage administration induced prenatal stress. Cell cultures were obtained from the brains of 1-day-old pups. Prenatal stress caused a disturbance of sensorimotor function in pups. Prenatal stress also produced alterations in the glial cultures, specifically, an increased percentage of microglia in the mixed glial cultures and an increased percentage of dead cells. Moreover, increased levels of IL1-β, TNF-α, NO, and an increased expression of CX3CR1 mRNA were found in microglia. However, the ratio of Bax/Bcl2 mRNA was reduced. Prenatal stress increased the vulnerability of microglia to lipopolysaccharide (LPS). The mixed glial culture derived from pups exposed to IMI showed greater morphological changes and the highest percentage of microglia. Microglia were characterized by the largest increase in the production of pro-inflammatory cytokines and NO, and the greatest reduction in the expression of CX3CR1 mRNA. Exposure to IMI reduced the effects of LPS on IL-1β production and Bax/Bcl2 mRNA, and exacerbated the effects of LPS on CX3CR1 mRNA expression. Prenatal administration of VEN induced protective effects on microglia, as measured by all studied parameters. Taken together, our data suggest that, by disturbing microglia function, exposure to even mild forms of chronic prenatal stress may predispose individuals to psychiatric or neurodevelopmental disorders. These data also indicate that chronic mild stress sensitizes microglia to immune challenges, which may lead to enhanced neuronal damage in the embryonic brain. The observed detrimental effects of IMI on microglial activity under conditions of prenatal stress may help to explain the teratogenic effects of IMI reported in the literature.

5-aminoisoquinolinone attenuates social behavior deficits and immune abnormalities in the BTBR T+ Itpr3tf/J mouse model for autism

Autism spectrum disorder (ASD) is diagnosed by core symptoms including impaired social communication and the presence of repetitive and stereotypical behaviors. There is also evidence for immune dysfunction in individuals with ASD, but it is a disease that is still insufficiently controlled by current treatment strategies. The use of 5-aminoisoquinolinone (5-AIQ) ameliorates several immune-mediated symptoms including rheumatoid arthritis and colitis, and has neuroprotective properties; however, its role in ASD is not yet characterized. In this study, we investigated the effect of 5-AIQ on sociability tests, self-grooming, marble burying, and locomotor activities in BTBR T+ Itpr3tf/J (BTBR) mice, which serve as an ASD animal model. We further investigated the possible molecular mechanism of 5-AIQ administration on CXCR4-, CXCR6-, IFN-γ-, IL-22-, NOS2-, STAT1-, T-bet-, and RORγT-producing CD3+ T cells isolated from the spleens of treated mice. We also explored its effects on mRNA expression in brain tissue. Our results showed that in BTBR mice, 5-AIQ treatment significantly prevented self-grooming and marble burying behaviors and enhanced social interactions without any adverse effects on locomotor activity/anxiety level. Additionally, 5-AIQ treatment substantially decreased CXCR4-, CXCR6-, IFN-γ-, IL-22-, NOS2-, STAT1-, T-bet-, and RORγT-producing CD3+ T cells in the spleen. Furthermore, 5-AIQ treatment decreased CXCR4, IFN-γ, IL-22, STAT1, and RORγT mRNA expression levels in brain tissue. Our findings demonstrated that 5-AIQ improved behavioral and immune abnormalities associated with ASD, which supports the hypothesis that 5-AIQ has important therapeutic potential for the treatment of behavioral and neuroimmune dysfunctions in ASD.

Macrophage migration inhibitory factor is differentially expressed in normal and choriocarcinoma trophoblast cells.

Trophoblast cells are specific for placenta, the organ necessary for development of the fetus. Trophoblast derived choriocarcinoma is a rare cancer, with high metastatic potential, invading surrounding tissues and distant organs. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine involved in a wide range of biological processes, which is increased in almost all human cancers. Expression of MIF in normal and choriocarcinoma trophoblast cells is investigated here, using normal extravillous trophoblast derived cell line HTR-8/SVneo, and choriocarcinoma cell lines JAR and JEG3. Expression of MIF and its receptors CD74 and CXCR2 was investigated at mRNA level using qPCR. Expression of MIF protein was studied using immunofluorescence and western blot, under reducing and native conditions, in whole cell lysates, subcellular fractions and conditioned media. The expression of MIF mRNA was similar in all three cell lines, while CD74 mRNA was more expressed in choriocarcinoma cells (14-fold for JAR, 12-fold for JEG3, p<0.01). CXCR2 mRNA was higher in JEG3 cell line compared to HTR-8/SVneo cells (6-fold, p<0.01). While the cellular level of MIF was similar, the level of secreted MIF was lower in JAR cell conditioned media compared to media of both HTR-8/SVneo (2.8-fold, p<0.01) and JEG3 cells (4.1-fold, p<0.001). Cellular distribution of MIF was similar between the studied cell types. MIF was predominantly cytoplasmic, but also detected in membrane, nuclear soluble and nuclear chromatin fraction. MIF appeared in high molecular weight complexes of >150 kDa under native conditions. A band of 140-145 kDa was consistently present in JEG3 cell lysates, while it was absent or very weak in other cell types. These results show that MIF/CD74 axis is shifted in choriocarcinoma, as previously shown for other cancers, and further justifies research towards the most effective MIF targeting therapeutics.

1063: Endometrial and myometrial cell CXCL12 expression in response to mechanical injury: implications for placenta accreta.

In view of increasing incidence of placenta accreta spectrum disorders, there is an urge to better understand the molecular mechanisms of abnormal trophoblast invasion. CXCL12 and its receptor, CXCR4, have been implicated in many physiologic and pathologic processes including cell migration and trafficking, tissue healing in the setting of mechanical, ischemic and inflammatory damage and cancer metastasis. CXCL12 functions as a chemokine, attracting cells expressing CXCR4. Prior studies demonstrate that CXCL12 is produced by endometrium while CXCR4 is present on trophoblast cells. In this study, we sought to assess if tissue injury would affect the expression of CXCL12 in endometrial and myometrial cells as well as CXCR4 expression in trophoblast cells using an in vitro model. Cultured primary human endometrial stromal and myometrial cells were subjected to a scratch injury. Cells were collected 4 hours after injury and CXCL12 mRNA levels in injured and non-injured cells were assessed using RT-qPCR. CXCR4 mRNA levels were assessed in 2 human first-trimester trophoblast cell lines and in primary cytotrophoblast from term placentas with RT-qPCR. Baseline levels of CXCL12 mRNA were higher in myometrial than endometrial stromal cell (2-ΔCt; 0.076 vs 0.011 respectively, p< 0.01). Scratch injury resulted in significant increase in CXCL12 mRNA levels in endometrial stromal (2-ΔCt; 0.048 from 0.011, p=0.02) but not in myometrial cells (2-ΔCt; 0.076 vs 0.069, p >0.05). CXCR4 mRNA was expressed at high levels in cytotrophoblast from term placentas, but not in the established trophoblast cell lines. Injury results in enhanced expression of CXCL12 in cultured endometrial stromal cells. CXCL12 secretion will attract the CXCR4 expressing trophoblasts. These results suggest that activation of this axis as part of the normal healing process is likely a factor modulating the increased invasiveness of the trophoblast in the setting of uterine injury. Modulation of CXCL12 expression or use of CXCR4 antagonists may prevent excessive placental invasion associated with placenta accreta.

Modulation of CXC-motif chemokine receptor 7, but not 4, expression is related to migration of the human prostate cancer cell LNCaP: regulation by androgen and inflammatory stimuli.

To elucidate the regulation, function of the chemokine CXC-motif ligand 12 (CXCL12) and its receptors (CXCR) 4 and 7 in prostate cancer tumor microenvironment. In-silico-analysis of expression in prostate cancer tissues. In-vitro comparison, testing of regulation in human prostate cancer cells LNCaP, DU145, and PC3. Dihydrotestosterone (DHT) treatments (0-10nM) were for 0-48h. The inflammatory agent Flagellin treatment (20ng/ml) was for 2h. Migration assays were performed for 24h using 10ng/ml CXCL12. Real-time PCR, western analysis, and migration assays were used to determine mRNA, protein, and functional changes, respectively. Malignant prostate cancer tissues exhibit higher CXCR4/7 mRNA ratio, and higher CXCR7 mRNA levels were detected in the androgen-responsive LNCaP cells. Putative androgen-responsive elements were identified in CXCR4, 7 gene, and exposure to DHT, flagellin increased CXCR4 mRNA but decreased CXCR7 mRNA levels in LNCaP cells. Androgen receptor siRNA significantly attenuated the effects of DHT on CXCR4, 7 mRNA in LNCaP cells. However, DHT and flagellin only decrease CXCR7 protein and additively increased migration of LNCaP cells towards CXCL12. Down regulation of CXCR7 protein by DHT and flagellin increased migration, supporting CXCR7 as decoy receptor counteracting CXCL12/CXCR4-mediated migration in prostate cancer cells.

Cardiac Differentiation of Adipose Tissue-Derived Stem Cells Is Driven by BMP4 and bFGF but Counteracted by 5-Azacytidine and Valproic Acid.

Objective Bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) play important roles in embryonic heart development. Also, two epigenetic modifying molecules, 5ˊ-azacytidine (5ˊ-Aza) and valproic acid (VPA) induce cardiomyogenesis in the infarcted heart. In this study, we first evaluated the role of BMP4 and bFGF in cardiac trans-differentiation and then the effectiveness of 5´-Aza and VPA in reprogramming and cardiac differentiation of human adipose tissue-derived stem cells (ADSCs). Materials and Methods In this experimental study, human ADSCs were isolated by collagenase I digestion. For cardiac differentiation, third to fifth-passaged ADSCs were treated with BMP4 alone or a combination of BMP4 and bFGF with or without 5ˊ-Aza and VPA pre-treatment. After 21 days, the expression of cardiac-specific markers was evaluated by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, immunocytochemistry, flow cytometry and western blot analyses. Results BMP4 and more prominently a combination of BMP4 and bFGF induced cardiac differentiation of human ADSCs. Epigenetic modification of the ADSCs by 5ˊ-Aza and VPA significantly upregulated the expression of OCT4A, SOX2, NANOG, Brachyury/T and GATA4 but downregulated GSC and NES mRNAs. Furthermore, pre-treatment with 5ˊ-Aza and VPA upregulated the expression of TBX5, ANF, CX43 and CXCR4 mRNAs in three-week differentiated ADSCs but downregulated the expression of some cardiac-specific genes and decreased the population of cardiac troponin I-expressing cells. ConclusionOur findings demonstrated the inductive role of BMP4 and especially BMP4 and bFGF combination in cardiac trans-differentiation of human ADSCs. Treatment with 5ˊ-Aza and VPA reprogrammed ADSCs toward a more pluripotent state and increased tendency of the ADSCs for mesodermal differentiation. Although pre-treatment with 5ˊ-Aza and VPA counteracted the cardiogenic effects of BMP4 and bFGF, it may be in favor of migration, engraftment and survival of the ADSCs after transplantation.

Carboxypeptidase E-∆N Promotes Proliferation and Invasion of Pancreatic Cancer Cells via Upregulation of CXCR2 Gene Expression.

Pancreatic cancer is one of the leading causes of cancer-related mortality worldwide. The molecular basis for the pathogenesis of this disease remains elusive. In this study, we have investigated the role of wild-type Carboxypeptidase E (CPE-WT) and a 40 kDa N-terminal truncated isoform, CPE-ΔN in promoting proliferation and invasion of Panc-1 cells, a pancreatic cancer cell line. Both CPE-WT and CPE-ΔN were expressed in Panc-1 and BXPC-3 pancreatic cancer cells. Immunocytochemical studies revealed that in CPE transfected Panc-1 cells, CPE-ΔN was found primarily in the nucleus, whereas CPE-WT was present exclusively in the cytoplasm as puncta, characteristic of secretory vesicles. Endogenous CPE-WT was secreted into the media. Overexpression of CPE-ΔN in Panc-1 cells resulted in enhancement of proliferation and invasion of these cells, as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay and Matrigel invasion assay, respectively. In contrast, the expression of CPE-WT protein at comparable levels to CPE-ΔN in Panc-1 cells resulted in promotion of proliferation but not invasion. Importantly, there was an upregulation of the expression of CXCR2 mRNA and protein in Panc-1 cells overexpressing CPE-ΔN, and these cells exhibited significant increase in proliferation in a CXCR2-dependent manner. Thus, CPE-ΔN may play an important role in promoting pancreatic cancer growth and malignancy through upregulating the expression of the metastasis-related gene, CXCR2.

Cxcr4 low FLT3/ITD+ Cells Exhibit Enhanced Resistance to the FLT3 Inhibitor Quizartinib Compared to Cxcr4 high FLT3/ITD+ Cells By Elevating Runx1 Expression

The Runx1 transcriptional factor is significantly elevated in human AML cells with FLT3/ITD mutations (Behrens et al. JEM 2017) and enhances the resistance of FLT3/ITD+ 32D cells to the type II FLT3 inhibitor quizartinib (Hirade et al. IJH 2016). While Cxcr4 levels are significantly downregulated in human FLT3/ITD+ AML cells and FLT3/ITD+ Ba/F3 cells compared to FLT3/ITD- cells (Onishi et al. JBC 2014), Runx1 transactivates Cxcr4 expression (Jacob et al. Blood 2010), indicating that Cxcr4 is inversely regulated by FLT3/ITD and Runx1. Studies have indicated that Cxcl12 in the microenvironment protects FLT3/ITD+AML cells from chemotherapeutic insults through Cxcr4 (Zheng et al. Blood 2009). Herein, we investigated the interaction between Cxcl12/Cxcr4 signaling and Runx1 on the refractory activity of FLT3/ITD+ cells against quizartinib. Runx1 shRNAs downregulated Cxcr4 levels in FLT3/ITD+ Ba/F3 cells, indicating that Runx1 enhances the expression of Cxcr4. However, a comparison of the intracellular Runx1 levels in FLT3/ITD+ Ba/F3 cells expressing different surface Cxcr4 levels that were sorted by FACS based on Cxcr4 expression demonstrated that the Runx1 protein levels were the highest in FLT3/ITD+ Ba/F3 cells harboring the lowest Cxcr4 compared to those expressing higher Cxcr4 levels, and were inversely correlated with the Cxcr4 levels (r=-0.78, P&lt;0.05, N=6). Similarly, Runx1 mRNA levels were negatively correlated with the Cxcr4 mRNA levels in human FLT3/ITD+ AML cells (r=-0.34, P&lt;0.01, N=78, GSE 1159), which was not observed in FLT3/ITD- AML cells (N=187). Since Cxcr4 expression is regulated by FLT3/ITD and Runx1, we next compared the response to quizartinib of FLT3/ITD+ Ba/F3 cells with different surface expression levels of Cxcr4 in the presence or absence of Cxcl12. No significant difference in proliferation was observed in cells cultured without quizartinib. By contrast, the number of viable cells incubated with quizartinib (2 or 5nM) was significantly higher in Cxcr4 dim cells compared to Cxcr4 intermediate and Cxcr4 high cells. Cxcl12 dose-dependently inhibited the reduction of viable cells induced by 5nM quizartinib in Cxcr4 dim FLT3/ITD+ Ba/F3 cells (N=5, P&lt;0.05). Similarly, 100ng/ml of Cxcl12 increased the number of Cxcr4 dim FLT3/ITD+ Ba/F3 cells that became refractory quizartinib (N=3, P&lt;0.05). Likewise, Cxcl12 dose dependently prevented the decline of the number of human FLT3/ITD+ MV4-11 and MOLM13 AML cells induced by quizartinib (N=3, P&lt;0.05). The prosurvival effect by Cxcl12 in Cxcr4 dim FLT3/ITD+ Ba/F3 cells was coincident with the Cxcl12-mediated elevation of Runx1 mRNA; however, Runx1 elevation after stimulation with 100ng/ml Cxcl12 was higher in Cxcr4 dim cells compared to Cxcr4 high cells. Runx1 shRNA significantly decreased proliferation and partially abrogated the anti-apoptotic effect of Cxcl12 against quizartinib in Cxcr4 dim FLT3/ITD+ Ba/F3 cells that were sensitive and refractory to quizartinib. ShRNA-mediated Runx1 knockdown also diminished chemotaxis towards Cxcl12 in quizartinib refractory Cxcr4 dim FLT3/ITD+ Ba/F3 cells (N=3, P&lt;0.05), concomitant with a reduction in Cxcr4 expression. By contrast, similar cyto-protective effects of 10-100ng/ml Cxcl12 against 5nM quizartinib were not observed in Cxcr4 high FLT3/ITD+ cells. Cxcl12 rather enhanced the proapoptotic effect of 5nM quizartinib in Cxcr4 high cells (N=5, P&lt;0.05). These data suggest that Cxcr4 dim FLT3/ITD+ cells are more refractory to quizartinib than Cxcr4 high cells, which is mediated by the elevation of Runx1. Studies have indicated that Cxcr4 levels are downregulated and Runx1 levels are upregulated by FLT3/ITD, even though Runx1 enhances Cxcr4 expression. Our data indicate that Cxcr4 dim FLT3/ITD+ cells are more refractory to quizartinib in the presence or absence of Cxcl12 concomitant with higher Runx1 levels, compared to Cxcr4 high cells. Antagonizing Runx1 partially abrogated the refractory against quizartinib in Cxcr4 dim cells in the presence or absence of Cxcl12. These data implicate that Cxcr4 dim FLT3/ITD+ cells display increased FLT3/ITD activity compared to Cxcr4 high cells as a consequence of elevated Runx1 expression. Runx1 also enhanced chemotaxis of quizartinib resistant FLT3/ITD+ cells to Cxcl12. Targeting Runx1 represents an additional strategy to overcome FLT3 inhibitor refractory in FLT3/ITD+AML cells that are protected in the microenvironment. Disclosures No relevant conflicts of interest to declare.

Predicting Angiogenesis by Endothelial Progenitor Cells Relying on In-Vitro Function Assays and VEGFR-2 Expression Levels.

Clinical trials have demonstrated the safety and efficacy of autologous endothelial progenitor cell (EPC) therapy in various diseases. Since EPCs’ functions are influenced by genetic, systemic and environmental factors, the therapeutic potential of each individual EPCs is unknown and may affect treatment outcome. Therefore, our aim was to compare EPCs function among healthy donors in order to predict blood vessel formation (angiogenesis) before autologous EPC transplantation. Human EPCs were isolated from the blood of ten volunteers. EPCs proliferation rate, chemoattractant ability, and CXCR4 mRNA levels were different among donors (p < 0.0001, p < 0.01, p < 0.001, respectively). A positive correlation was found between SDF-1, CXCR4, and EPCs proliferation (R = 0.736, p < 0.05 and R = 0.8, p < 0.01, respectively). In-vivo, blood vessels were counted ten days after EPCs transplantation in a subcutaneous mouse model. Mean vessel density was different among donors (p = 0.0001); nevertheless, donors with the lowest vessel densities were higher compared to control (p < 0.05). Finally, using a linear regression model, a mathematical equation was generated to predict blood vessel density relying on: (i) EPCs chemoattractivity, and (ii) VEGFR-2 mRNA levels. Results reveal differences in EPCs functions among healthy individuals, emphasizing the need for a potency assay to pave the way for standardized research and clinical use of human EPCs.

CX3CL1-CX3CR1 Axis: A New Player in Coeliac Disease Pathogenesis.

Background: The CX3CL1–CX3CR1 axis has been related to numerous diseases. The aim of our study was to investigate its involvement in coeliac disease (CD) pathogenesis, particularly in the early phase of the disease. Methods: We collected peripheral blood from CD patients and controls, enrolled in a 3-day gluten challenge, to study soluble CX3CL1, I-TAC and MIG by Luminex, CX3CL1 and CX3CR1 gene expression by qPCR, and CX3CR1 protein expression in monocytes and CD8+, CD4+ and γδ+ T cells, by flow cytometry. We also analysed the expression of the CX3CL1 and CX3CR1 mRNA and protein in the duodenal biopsies of CD patients with active and treated disease, and in non-CD control individuals, by qPCR and immunohistochemistry. Results: After the gluten challenge, increased levels of CX3CL1, I-TAC and MIG proteins were observed in the peripheral blood of CD patients, with no changes in CX3CL1 mRNA, or CX3CR1 mRNA and protein. Regarding duodenal tissue, CX3CL1 was absent or barely present in the superficial and basal epithelium of CD patients, contrasting with the moderate to high levels present in controls. Conclusions: CX3CL1 seems to be involved in the appearance and progression of CD, and it appears to be a potential diagnostic biomarker. Its use as an alternative therapeutic target in CD deserves further research.

Influence of β-D-mannuronic Acid, as a New Member of Non-steroidal Anti- Inflammatory Drugs Family, on the Expression Pattern of Chemokines and their Receptors in Rheumatoid Arthritis.

Based on the encouraging results of phase III clinical trial of β-Dmannuronic acid (M2000) (as a new anti-inflammatory drug) in patients with RA, in this study, we aimed to evaluate the effects of this drug on the expression of chemokines and their receptors in PBMCs of RA patients. PBMCs of RA patients and healthy controls were separated and the patients' cells were treated with low, moderate and high doses (5, 25 and 50 μg/mL) of M2000 and optimum dose (1 μg/mL) of diclofenac, as a control in RPMI-1640 medium. Real-time PCR was used for evaluating the mRNA expression of CXCR3, CXCR4, CCR2, CCR5 and CCL2/MCP-1. Cell surface expression of CCR2 was investigated using flow cytometry. CCR5 mRNA expression reduced significantly, after treatment of the patients' cells with all three doses of M2000 and optimum dose of diclofenac. CXCR3 mRNA expression was downregulated significantly followed by the treatment of these cells with moderate and high doses of M2000 and optimum dose of diclofenac. CXCR4 mRNA expression declined significantly after the treatment of these cells with moderate and high doses of M2000. CCL2 mRNA expression significantly reduced only followed by the treatment of these cells with a high dose of M2000, whereas, mRNA and cell surface expressions of CCR2 diminished significantly followed by the treatment of these cells with a high dose of M2000 and optimum dose of diclofenac. According to our results, M2000 through the down-regulation of chemokines and their receptors may restrict the infiltration of immune cells into the synovium.

455PD - First-in-human, first-in-class phase I study of MTL-CEBPA, a RNA oligonucleotide targeting the myeloid cell master regulator C/EBP-α, in patients with advanced hepatocellular cancer (HCC)

BackgroundMTL-CEBPA is a first-in-class small activating RNA (saRNA) oligonucleotide which specifically up-regulates the myeloid cell master regulator, C/EBP-α (CCAAT/enhancer-binding protein alpha). MethodsWe conducted a phase I, 3+3 dose escalation and dose expansion trial of MTL-CEBPA in adults with HCC or secondary liver cancer. Patients received intravenous MTL-CEBPA at 28-160mg/m2 for 3 weeks either QW, BIW at d1 and d2, BIW at d1 and d3, or TIW at d1, d2, and d3 followed by a rest period of 1 week. Adverse events (AEs), serum PK, WBC biomarkers and anti-tumour activity were assessed. Results38 participants (31 HCC, 4 colorectal, 2 fibrolamellar, 1 ampullary) have been treated across 6 dose levels (28-160mg/m2) and 3 dosing schedules: 29M/9F, median age 66 years (range 27 - 80), ECOG PS 0/1: 16/22. In 28 HCC patients evaluable for efficacy, PR was achieved in 1pt, SD>1 year in 1 patient and SD<1 year in 11 pts. After discontinuation of MTL-CEBPA, four sorafenib- naïve HCC patients were treated with sorafenib; 3/4 had durable CR (14, 12 and 9 months) and 1/4 had PR [6 months] by RECIST 1.1. Treatment-related adverse events (all grades) that occurred in more than 10% of patients were fatigue (23.7%), thrombocytopaenia (13.2%), anaemia (13.2%), elevated AST (13.2%), elevated ALP (10.5%), hypoalbuminaemia (10.5%), increased ALT (10.5%) and increased bilirubin (10.5%); no maximum dose was reached in any dosing cohorts. Target engagement was evidenced in evaluable patients by a significant 1.5 fold increase in CEBPA mRNA in WBC and a significant increase in WBC count consistent with C/EBP-α dependent granulopoiesis. Altered immunological markers were observed in WBC of one patient including decreased levels of ADA, PD-L1 and CXCR4 mRNA. ConclusionsMTL-CEBPA is a first-in-class therapy targeting the myeloid cell master regulator C/EBP-α. In HCC patients MTL-CEBPA demonstrated a good safety profile, induced altered gene expression in WBC and as well as anti-tumour activity. These encouraging phase I data validate targeting of CEBP-α and have prompted MTL-CEBPA + sorafenib combination studies in HCC. Clinical trial identificationNCT02716012. Legal entity responsible for the studyMiNA Therapeutics. FundingMiNA Therapeutics. DisclosureD. Sarker: Travel / Accommodation / Expenses: MiNA Therapeutics; Honoraria (self): MSD; Honoraria (self): EISAI; Honoraria (self): BAYER; Honoraria (self), Travel / Accommodation / Expenses: IPSEN. K. HUANG: Research grant / Funding (self): MiNA Therapeutics. N. Habib: Shareholder / Stockholder / Stock options: MiNA Therapeutics. All other authors have declared no conflicts of interest.