Necrotizing enterocolitis (NEC) is a critical neonatal disease with a high mortality. The possibility that miRNAs may play an important role in NEC has raised great attention. Hence, the present study identified biomarkers that affected NEC in newborn progression through miRNA and gene expression profile analysis. miRNA chip GSE68054 and gene chip GSE46619 of NEC in newborn were analyzed to screen out differentially expressed miRNA and differentially expressed genes (DEGs). Next, target genes of differentially expressed miRNA were predicted, and differentially expressed miRNA-DEG regulatory network was constructed to select key miRNAs. After gene ontology and kyoto encyclopedia of genes and genomes enrichment analysis on target genes of key miRNAs, the target genes enriched in pathways were extracted to establish differentially expressed miRNA-DEG-disease gene network for gene interaction analysis. Targetting relationship between miRNAs and target genes was verified. A total of 15 miRNAs were differentially expressed in NEC in newborn, amongst which miR-429/200a/b and miR-141/200c clusters were poorly expressed and might play a significant role in NEC in newborn. Besides, target genes of miR-429/200a/b and miR-141/200c clusters were enriched in 11 signaling pathways. Vascular endothelial growth factor (VEGFA), E-selectin (SELE), kinase insert domain receptor (KDR), fms-related tyrosine kinase 1 (FLT1), and hepatocyte growth factor (HGF) were highly expressed in NEC in newborn, which were negatively regulated by miR-429/200a/b and miR-141/200c clusters and shared close association with disease genes. miR-429/200a/b and miR-141/200c clusters are poorly expressed while their target genes (VEGFA, SELE, KDR, FLT1, and HGF) are highly expressed in NEC in newborn, which might be identified as important biomarkers for this disease.

BACKGROUND

Preeclampsia (PE) affects 5-8% of all pregnant women and can trigger a severe gestational hypertension framework and eventually develop into eclampsia and HELLP syndrome. Anticipating the damage would be important in order to establish procedures that can reduce adverse outcomes. For this reason, many researches are undertaken to identify ways to make a diagnosis of preeclampsia as early as possible. It has been highlighted in literature the study: the sFlt1 (soluble fms-like tyrosine kinase-1) has been implicated in the precocious diagnosis of pre eclampsia. The sFlt1 is an anti-angiogenic factor produced in response to oxidative stress derived from the deleterious effects of pre-eclampsia.

OBJECTIVE

The objective of the study was to evaluate the role of Soluble fms-like tyrosine kinase-1 in the diagnosis of preeclampsia.

METHODS

This is a review conducted in the database PubMed and Lilacs. For this purpose, we used the following MeSH, "Vascular Endothelial Growth Factor Receptor-1" OR "FLT1 protein, human" AND "Pre-Eclampsia/diagnosis" in PubMed and "Pre-eclampsia" AND "SFLT1A" in Lilacs, resulting in 84 papers. After reading the abstracts of these studies, we selected the articles analyzed taking into consideration the criteria for inclusion and exclusion. We excluded publications that were not in the period under study (2008 to July 2011) and by study design. Including only case-control, cohort and prospective observational. For a critical analysis of the material, we used the following indicators: researcher, years, central theme, participants, study design and primary outcome.

RESULTS

The final results of this study were composed of seven articles and are shown for each target outcome. These vary according to gestational age at which PE is installed and the marker studied (sFlt1 alone or its relation to PlGF - sFlt1/PIGF). Six studies showed greater levels of sFlt1 for the preeclampsia groups when compared to the control group. Significantly differences in antiangiogenic factors seric levels were not found among preeclamptic and eclamptic patients. When associated with another factor, like PIGF, a greater efficacy in the diagnosis of early preeclampsia is shown. Of the studies analyzed, only one (Lynch et al) showed no significant difference between the values of sFlt-1 in groups of early PE, late PE and control for gestational ages between 10 and 15 weeks. As for the relation sFlt-1/PIGF, five studies have considered it even better for PE diagnosis when compared to sFlt-1 isolated.

CONCLUSIONS

The dosage of sFlt1 may be a relevant resource for the early diagnosis of preeclampsia before the installation of target organ damage, especially if measured in the period between 12 and 28 weeks of gestational age. Whereas sFlt-1 manifests itself before the 20th week, that may be interesting clinical point of view since it is this phase that settles the most severe cases, when the adoption of care could prevent further risks. The relationship sFlt1/PIGF, was more appropriate than the measurement of sFlt1 alone. Additional studies are needed to: amplification of the number of women evaluated, establishing gestational age appropriate for study, serum standard and need to consider the relationship between sFlt1 and other factors pro and/or anti-angiogenic.

Background: Preeclampsia is a dangerous cardiovascular disorder of pregnancy that leads to an increased risk of future cardiovascular and metabolic disorders. Much of the pathogenesis and mechanisms involved in cardiac health in preeclampsia are unknown. A novel anti-angiogenic protein, FKBPL, is emerging as having a potential role in both preeclampsia and cardiovascular disease (CVD). Therefore, in this study we aimed to characterise cardiac health and FKBPL regulation in the rat reduced uterine perfusion pressure (RUPP) and a 3D cardiac spheroid model of preeclampsia.

Methods: The RUPP model was induced in pregnant rats and histological analysis performed on the heart, kidney, liver and placenta (n ≥ 6). Picrosirius red staining was performed to quantify collagen I and III deposition in rat hearts, placentae and livers as an indicator of fibrosis. RT-qPCR was used to determine changes in Fkbpl, Icam1, Vcam1, Flt1 and Vegfa mRNA in hearts and/or placentae and ELISA to evaluate cardiac brain natriuretic peptide (BNP45) and FKBPL secretion. Immunofluorescent staining was also conducted to analyse the expression of cardiac FKBPL. Cardiac spheroids were generated using human cardiac fibroblasts and human coronary artery endothelial cells and treated with patient plasma from normotensive controls, early-onset preeclampsia (EOPE) and late-onset preeclampsia (LOPE); n = 3. FKBPL and CD31 expression was quantified by immunofluorescent labelling.

Results: The RUPP procedure induced significant increases in blood pressure (p < 0.001), collagen deposition (p < 0.001) and cardiac BNP45 (p < 0.05). It also induced a significant increase in cardiac FKBPL mRNA (p < 0.05) and protein expression (p < 0.01). RUPP placentae also exhibited increased collagen deposition and decreased Flt1 mRNA expression (p < 0.05). RUPP kidneys revealed an increase in average glomerular size (p < 0.05). Cardiac spheroids showed a significant increase in FKBPL expression when treated with LOPE plasma (p < 0.05) and a trend towards increased FKBPL expression following treatment with EOPE plasma (p = 0.06).

Conclusions: The rat RUPP model induced cardiac, renal and placental features reflective of preeclampsia. FKBPL was increased in the hearts of RUPP rats and cardiac spheroids treated with plasma from women with preeclampsia, perhaps reflective of restricted angiogenesis and inflammation in this disorder. Elucidation of these novel FKBPL mechanisms in cardiac health in preeclampsia could be key in preventing future CVD.

Keywords: Cardiac spheroids; Cardiovascular disease; FKBPL; Preeclampsia; Reduced uterine perfusion pressure.

Coke oven emissions (COEs) are common particle pollutants in occupational environment and the major constituents of COEs are polycyclic aromatic hydrocarbons (PAHs). Previously, we identified aberrant methylation of the fms related tyrosine kinase 1 (FLT1) gene over the course of benzo(a)pyrene (BaP)-induced cell transformation via genome-wide methylation array. To quantify FLT1 methylation, we established a bisulfite pyrosequencing assay and examined the FLT1 hypermethylation in several human cancers. The results revealed that 70.0% (21/30 pairs) of lung cancers harbored hypermethylated FLT1 and concomitant suppression of gene expression compared to the adjacent tissues. This implies that FLT1 hypermethylation might play a role in malignant cell transformation. In addition, FLT1 hypermethylation and gene suppression appeared in primary human lymphocytes in a dose-response manner following COEs treatment. To explore whether FLT1 methylation is correlated with COEs exposure and DNA damage, we recruited 144 male subjects who had been exposed to high levels of COEs and 84 male control subjects. Notably, the FLT1 methylation in peripheral blood lymphocytes (PBLCs) of the COEs-exposed group (19.8 ± 3.2%) was enhanced by 17.9% compared to that of the control group (16.8 ± 2.8%) (P < 0.001). The FLT1 methylation status was positively correlated with urinary 1-hydroxypyrene (1-OHP) levels, an internal exposure marker of PAHs (β = 0.029, 95% CI = 0.010-0.048, P = 0.003) and positively correlated with DNA damage (β_OTM = 0.024, 95% CI = 0.007-0.040, P = 0.005; β_{Tail DNA} = 0.035, 95% CI = 0.0017-0.054, P < 0.001) indicated by comet assay. Taken together, these findings indicate that FLT1 might be a tumor suppressor, and its hypermethylation might contribute to PAHs-induced carcinogenicity.

In normal pregnancy, the soluble form of FMS-like tyrosine kinase-1 (sFLT1)/ vascular endothelial growth factor receptor-1 (sVEGFR-1), a VEGF-trapping protein, is expressed in trophoblasts of the placenta, suggesting that it plays an important role in the physiological barrier between fetal and maternal angiogenesis, when stimulated with VEGF-A. In pathological conditions such as preeclampsia (PE), sFLT1 protein is abnormally overexpressed in trophoblasts and secreted into the serum, which could cause hypertension and proteinuria on the maternal side and growth retardation on the fetal side. Detection of an abnormal increase in serum sFLT1 during the early to middle stages of PE is essential for proper initiation of medical care. To carry out this screening for sFLT1, we developed an easier and relatively low-cost sandwich-type ELISA method using a single mixture of human serum sample with an anti-FLT1 antibody and heparin-beads, namely heparin-beads-coupled ELISA (HB-ELISA). This method takes only about 2 h, and the sFLT1 values were similar levels with commercially available recent ELISA kits: the serum sFLT1 protein was approximately 4.3-fold increased in severe PE compared with those in normal pregnancy.

A novel, non-terminal surgical procedure to remove a single placentome from the pregnant ewe for gene expression and histological analyses was recently developed in our laboratory. This technique allows for evaluation of nutritional insults on placental development at more than one stage of gestation using a single animal. Early attempts to develop a similar technique in cattle were met with complications due to inaccessibility of the gravid uterine horn because of its location and mass. One alternative is to collect a placentome from the contralateral uterine horn; however, the question remains as to whether gene expression varies among placentomes based on location relative to the fetus. Pregnant heifers were maintained on forage during early gestation and later moved into pens with a Calan gate system (American Calan, Northwood, NH). On gestational day (GD) 158, five heifers were assigned to receive a hay-based diet formulated to meet 100% of NRC requirements, and five heifers were fed 70% of NRC requirements until necropsy on GD244. At necropsy, a single representative placentome was selected for analysis from the antimesometrial side: (1) of the gravid uterine horn central to the amnion, (2) over the allantois immediately adjacent to the amnion, (3) in the tip of the gravid uterine horn, and (4) in the tip of the contralateral uterine horn. Mean placentome weight was greater (P < 0.05) for locations central to the amnion and allantois compared to locations within the tips of the ipsilateral and contralateral horns, respectively. Gene expression for angiogenic factors (FGF2, ODC1, VEGFA, and FLT1), nutrient transporters (SLC7A1 and SLC2A1), and factors associated with hormone action (ESR1, IGF1, IGFBP3, CSH1, and PAG1) were unaffected (P > 0.05) by dietary treatment or location of the placentome. Results indicate that location of the placentome in relation to the fetus does not impact gene expression, enhancing the efficacy of nonterminal methodologies for sampling gene expression in placentomes.

Keywords: bovine; gene expression; nutrient restriction; placentome.

OBJECTIVE

To study the expression regularity of vascular endothelial growth factor (VEGF) during the process of fracture healing, and the type of VEGF receptor expressed in the vascular endothelial cells of the fracture site.

METHODS

The fracture model was made in the middle part of left radius in 35 rabbits. The specimens from the fracture site were harvested at 8, 24, 72 hours and 1, 3, 5, 8 weeks, and then fixed, decalcified, and sectioned frozenly to detect the expression of VEGF and its receptor at the fracture site by in situ hybridization and immunochemical assays.

RESULTS

VEGF mRNA and VEGF expression was detected in many kinds of cells at the fracture site during 8 hours to 8 weeks after fracture. Flt1 receptor of VEGF was found in the vascular endothelial cells at the fracture site during 8 hours to 8 weeks after fracture, and strong expression of flk1 receptor was detected from 3 days to 3 weeks after fracture.

CONCLUSIONS

The expression of VEGF and flt1 receptor appears during the whole course of fracture healing, especially from 1 to 3 weeks. Flk1 receptor is highly expressed in a definite period after fracture. VEGF is proved to be involved in the vascular reconstruction and fracture healing.

This article describes a screening platform to identify compounds that affect human embryonic vascular development. The procedure comprises the generation of human embryonic-like endothelial cells (ECs) from human pluripotent stem cells (hPSCs) and subsequent maturation under arterial flow conditions; the use of these cells for the high-throughput screening of small molecules that specifically inhibit the survival of embryonic-like ECs; the confirmation of the hits in embryonic-like ECs cultured under flow shear stress; and final validation in mouse embryonic ECs. The embryonic-like ECs express embryonic genes including DLL1, EPHB2, LYN, TEK, ID1, NRP2, CAST, FLT1, IGF1, DKK3, NIN, LEF1, and SORBS3. The entire screening procedure (without the validation step) can be completed within 1 month. This platform is an alternative/complement to standard animal protocols for assessing the effects of chemicals on embryonic vascular development. © 2019 by John Wiley & Sons, Inc.

The potential angiogenic effect of roxarsone, a feed additive widely used to promote animal growth worldwide, was demonstrated recently. We explored the mechanism of vascular endothelial growth factor (VEGF) and its receptor (VEGFR) in roxarsone promotion of rat vascular endothelial cells (ECs) and B16F10 mouse xenografts. ECs were treated with 0.1-50 μM roxarsone or with roxarsone plus 10 ng/mL VEGF, VEGFR1 (Flt1), or VEGFR2 (Flk1) antibodies for 12-48 h to examine their role in cell growth promotion. Small interfering RNA (siRNA) targeting Vegf, Flt1, and Flk1 were transfected in the ECs, and we measured the expression level, cell proliferation, migration, and tube formation ability. The siRNA targeting Vegf or Flk1 were injected intratumorally in the B16F10 xenografts of mice that received 25 mg/kg roxarsone orally. Cell viability and VEGF expression following roxarsone treatment were significantly higher than that of the control (P < 0.05), peaking following treatment with 1.0 μM roxarsone. Compared to roxarsone alone, the VEGF antibody decreased cell promotion by roxarsone (P < 0.05), and the Flk1 antibody greatly reduced cell viability compared to the Flt1 antibody (P < 0.01). Roxarsone and Flk1 antibody co-treatment increased supernatant VEGF significantly, while cellular VEGF was obviously decreased (P < 0.01), whereas there was no significant difference following Flt1 antibody blockade. The siRNA against Vegf or Flk1 significantly attenuated the roxarsone promotion effects on EC proliferation, migration, and tube-like formation (P < 0.01), whereas the siRNA against Flt1 effected no obvious differences. Furthermore, the RNA interference significantly weakened the roxarsone-induced increase in xenograft weight and volume, and VEGF and Flk1 expression. Roxarsone promotion of rat EC growth, migration, and tube-like formation in vitro and of B16F10 mouse xenograft model tumor growth and angiogenesis involves a VEGF/Flk1 mechanism.

Chlorpropham is used to prevent sprouting in stored agricultural products. It functions through mitosis inhibition or microtubule assembly inhibition in target organisms including plants, protozoa, and fungi. Although the toxicity ranges of chlorpropham in different organisms are known, specific studies on the environmental contamination and the harmful effects of chlorpropham has not been elucidated. In the present study, we demonstrated that toxicity assays of chlorpropham using zebrafish embryos showed pathological morphology alteration with half the embryos undergoing embryonic death. Fluorescent dye was used in live embryos to identify whether oxidative stress and apoptosis mediated developmental malformation. Specific genes related to apoptosis, ccnd1, ccne1, and cdk6, belonging to cell cycle regulation were downregulated on exposure to sublethal concentrations of chlorpropham. Moreover, vascular morphogenesis, which contributes to the cardiovascular circulatory system, was disrupted by chlorpropham along with decreased expression of specific regulators (flt1, kdr, and vegfaa). These data suggest that environmentally preserved chlorpropham is a potential pollutant in non-target species, especially in aquatic organisms, and emphasizes the need for caution regarding the ecotoxicity of chlorpropham.

Keywords: Apoptosis; Chlorpropham; Developmental toxicity; Oxidative stress; Vascular morphogenesis; Zebrafish embryos.

SummaryIn eutherian mammals, the placenta plays a critical role in embryo development by supplying nutrients and hormones and mediating interaction with the mother. To establish the fine connection between mother and embryo, the placenta needs to be formed normally, but the mechanism of placental differentiation is not fully understood. We previously revealed that mouse prolyl oligopeptidase (POP) plays a role in trophoblast stem cell (TSC) differentiation into two placental cell types, spongiotrophoblasts (SpT) and trophoblast giant cells. Here, we focused on SpT differentiation and attempted to elucidate a molecular mechanism. For Ascl2, Arnt, and Egfr genes that are indispensable for SpT formation, we found that a POP-specific inhibitor, SUAM-14746, significantly decreased Ascl2 expression, which was consistent with a significant decrease in expression of Flt1, a gene downstream of Ascl2. Although this downregulation was unlikely to be mediated by the PI3K-Akt pathway, our results indicated that POP controls TSC differentiation into SpT by regulating the Ascl2 gene.

Vascular endothelial growth factor-A (VEGF-A) is a principal regulator of hematopoiesis as well as angiogenesis. However, the functions of VEGF-A and its receptors (VEGFRs) in the differentiation of mast cells (MCs) in the skin remain unclear. The aim of this study was to determine the expression patterns of two VEGFRs (Flk1 and Flt1) in the skin MCs during development and maturation in rats. From the 17th days of embryonic development (E17) to 1 day after birth (Day 1), most of skin MCs were immature cells containing predominant alcian blue (AB)⁺ rather than safranin O (SO)⁺ granules (AB>SO MCs). AB>SO MC proportions gradually decreased, while mature AB<SO MC proportions increased from Day 7 to 28. Flk1⁺ MC proportions increased from E20 and reached to approximately 90% from Day 1 to 21, thereafter decreased to about 10% at Day 60 and 90. Flk1⁺ MC proportions changed almost in parallel with the numbers of MCs and Ki67⁺ MC proportions from E17 to Day 90. The proportions of MCs with both nuclear and cytoplasmic Flt1-immunoreactivity were markedly increased at Day 28, when the proportions of nuclear Flk1⁺, Ki67⁺, and AB>SO MCs had significantly decreased, and AB<SO MC proportions significantly increased. Considering that the main function of Flt1 is suppression of Flk1 effects, our results indicated that cross-talk between Flk1 and Flt1 regulates the proliferation and maturation of the skin MCs during late embryonic and neonatal development in rats.

Imprinted genes in which only one of the two parental chromosome copies is expressed have a substantial effect on mammalian ontogenesis. On mouse distal chromosome 7, the paternally expressed gene insulin-like growth factor 2 (Igf2) is separated by approximate 100 kb from the maternally expressed non-coding gene H19. However, there is limited knowledge of the manner in which Igf2 transcription affects the other genes involved in embryonic development. To clarify this, we performed quantitative gene expression analysis for representative angiogenic factors-Vegf, Flt1, Flt4, Flk1, Ang1, Ang2, Tie1, and Tie2-for 3 types of bi-maternal conceptuses containing genomes with non-growing (ng) and fully grown (fg) oocytes. The genetic backgrounds of the ng oocytes were 1) the wild type (ng(wt)), 2) mutant mice carrying a 3-kb deletion of the H19 transcription unit (ng(H19Delta3-KO)/fg) and 3) mutant mice carrying a 13-kb deletion in the H19 transcription unit, including the germline-derived differentially methylated region on chromosome 7 (ng(H19Delta13-KO)/fg). In the ng(wt)/fg and ng(H19Delta3-KO)/fg placentae, Vegf and Flt1 were upregulated compared with the mean value for the wt placenta, whereas in the ng(H19Delta13-KO)/fg placenta, these transcriptional levels were restored. In the fetus, however, only 2 genes among the 8 genes analyzed were significantly changed in the bi-maternal fetuses, indicating that the effects of the Igf2 mRNA level on angiogenic factor transcription in the fetus differed from those in the placenta. Our results indicated that the Igf2 mRNA level affects transcription of angiogenic factors in both bi-maternal placentae and fetuses.

Background: Bacillus Calmette-Guérin (BCG) immunotherapy, the standard adjuvant intravesical therapy for some intermediate and most high-risk non-muscle invasive bladder cancers (NMIBCs), suffers from a heterogenous response rate. Molecular markers to help guide responses are scarce and currently not used in the clinical setting.

Methods: To identify novel biomarkers and pathways involved in response to BCG immunotherapy, we performed a genome-wide DNA methylation analysis of NMIBCs before BCG therapy. Genome-wide DNA methylation profiles of DNA isolated from tumors of 26 BCG responders and 27 failures were obtained using the Infinium MethylationEPIC BeadChip.

Results: Distinct DNA methylation patterns were found by genome-wide analysis in the two groups. Differentially methylated CpG sites were predominantly located in gene promoters and gene bodies associated with bacterial invasion of epithelial cells, chemokine signaling, endocytosis, and focal adhesion. In total, 40 genomic regions with a significant difference in methylation between responders and failures were detected. The differential methylation state of six of these regions, localized in the promoters of the genes GPR158, KLF8, C12orf42, WDR44, FLT1, and CHST11, were internally validated by bisulfite-sequencing. GPR158 promoter hypermethylation was the best predictor of BCG failure with an AUC of 0.809 (p-value < 0.001).

Conclusions: Tumors from BCG responders and BCG failures harbor distinct DNA methylation profiles. Differentially methylated DNA regions were detected in genes related to pathways involved in bacterial invasion of cells or focal adhesion. We identified candidate DNA methylation biomarkers that may help to predict patient prognosis after external validation in larger, well-designed cohorts.

Keywords: BCG refractory; Bacillus Calmette-Guérin; DNA methylation marker; Illumina MethylationEPIC BeadChip; bladder cancer; high-risk bladder cancer; urothelial cancer.

BACKGROUND

Data on genetic variants that can predict follow-up cardiovascular events are highly limited, particularly for Asians. The aim of this study was to validate the effects of two variants in FLT1 and 9p21 on long-term cardiovascular outcomes in high-risk Korean patients.

METHODS

We examined the prognostic values of the rs9508025 and rs1333049 variants that were found to be associated with coronary artery disease (CAD) risk in a previous Korean genome-wide association study. A total of 2693 patients (mean age: 55.2 years; male: 55.2%) with CAD or its risk factors at baseline were enrolled and followed for major adverse cardiac events (MACE).

RESULTS

During the mean follow-up of 8.8 years, 15.4% of the patients experienced MACE. Kaplan-Meier curves showed that MACE-free survival was different according to the genotype of rs9508025 (log rank p = 0.02), whereas rs1333049 genotype did not correlate with the prognosis. Multivariate Cox proportional hazard analysis showed that C-allele of rs9508025 was significantly associated with a high rate of MACE, while rs1333049 was not. Further analyses demonstrated that the association of the rs9508025 variant with MACE was mainly due to its relation to coronary revascularization, which was also associated with the rs1333049 variant. In an additional analysis, rs9508025 was found to be an independent determinant of the outcome only in the subgroup with history of CAD.

CONCLUSIONS

rs9508025 in FLT1 was significantly associated with long-term cardiovascular events, particularly in patients with prior CAD. The association of rs1333049 in 9p21 was not significant.

Background: Podocyte function is tightly linked to the complex organization of its cytoskeleton, and adhesion to the underlying glomerular basement membrane. Adhesion of cultured podocytes to a variety of substrates is reported to correlate with podocyte health. Methods: To identify novel genes that are important for podocyte function, we designed an in vitrogenetic screen based on podocyte adhesion to plates coated with either fibronectin or soluble FLT1/Fc. Results: A genome-scale pooled RNA interference screen on immortalized human podocytes identified 77 genes that increased adhesion to fibronectin, 101 genes that increased adhesion to sFLT1/Fc, and 44 genes that increased adhesion to both substrates when knocked down. Multiple shRNAs against each of DPH1, DPH2, DPH3, and DPH4were top hits for increased adhesion. Immortalized human podocyte cells stably expressing these hairpins displayed increased adhesion to both substrates. We then used CRISPR-Cas9 to generate podocyte knockout cells for DPH1, DPH2,or DPH3which also displayed increased adhesion to both fibronectin and sFLT1/Fc, as well as a spreading defect. Last, we showed that Drosophila nephrocyte-specific knock-down of Dph1, Dph2, and Dph4results in altered nephrocyte function. Conclusions: In summary, we report a novel high-throughput method to identify genes important for podocyte function. Given the central role of podocyte adhesion as a marker of podocyte health, these data are a rich source of candidate regulators of glomerular disease.

Transmembrane 2 (TMEM2) gene inhibits chronic hepatitis-B virus (HBV) infection, while the underlying molecular mechanisms remain unknown. Transcriptome alterations in HepG2 cells following TMEM2 overexpression or silencing by shRNA were analyzed by next-generation sequencing. Both overexpression and knockdown of the TMEM2 gene caused wide-spread changes in gene expression in HepG2 cells. Differentially expressed genes caused by altered TMEM2 gene expression were associated with multiple biological processes linked with viral infection and various signaling pathways. KEGG analysis revealed that many of the differentially expressed genes were enriched in the PI3K/AKT signaling pathway. Moreover, we show that genes related to the PI3K/AKT signaling pathway, such as SYK, FLT4, AKT3, FLT1, and IL6, are biological targets regulated by TMEM2 in HepG2 cells. This is the first transcriptome-wide study in which TMEM2-regulated genes in HepG2 cells have been screened. Our findings elucidate the molecular events associated with TMEM2-mediated hepatocyte pathogenesis in chronic HBV infection.

Background: In order to improve targeted therapeutic approaches for children with atopic dermatitis (AD), novel insights into the molecular mechanisms and environmental exposures that differentially contribute to disease phenotypes are required. We wished to identify AD immunological endotypes in South African children from rural and urban environments.

Methods: We measured immunological, socioeconomic and environmental factors in healthy children (n=74) and children with AD (n=78), in rural and urban settings from the same ethno-linguistic AmaXhosa background in South Africa.

Results: Circulating eosinophils, monocytes, TARC, MCP-4, IL-16 and allergen-specific IgE levels were elevated, while IL-17A and IL-23 levels were reduced, in children with AD regardless of their location. Independent of AD, children living in a rural environment had the highest levels of TNFα, TNFβ, IL-1α, IL-6, IL-8, IL-21, MCP-1, MIP-1α, MIP-1β, MDC, sICAM1, sVCAM1, VEGFA, VEGFD and Tie2, suggesting a generalized microinflammation or a pattern of trained immunity without any specific T_H polarization. In contrast, IL-15, IL-22, Flt1, PIGF and βFGF were highest in urban children. Rural healthy children had the lowest levels of food-allergen specific IgG4. Early life nutritional factors, medications, animal exposures, indoor environment, sunlight exposure, household size, household income and parental education levels were associated with differences in circulating cytokine levels.

Conclusions: This study highlights the immunological impact of environmental exposures and socio-economic status in the manifestation of immune endotypes in children with AD living in urban and rural areas, which are important in selecting appropriately matched immunological therapies for treatment of AD.

Keywords: Atopic dermatitis; cytokines; endotypes; environment; personalized medicine.

Advanced pancreatic cancer is one of the most lethal malignant human diseases lacking effective treatment. Its extremely low survival rate necessitates development of novel therapeutic approach. Human neural stem cells (NSCs) are known to have tumor-tropic effect. We genetically engineered them to express rabbit carboxyl esterase (F3.CE), which activates prodrug CPT-11(irinotecan) into potent metabolite SN-38. We found significant inhibition of the growth of BxPC3 human pancreatic cancer cell line in vitro by F3.CE in presence of CPT-11. Apoptosis was also markedly increased in BxPC3 cells treated with F3.CE and CPT-11. The ligand VEGF and receptor VEGF-1(Flt1) were identified to be the relevant tumor-tropic chemoattractant. We confirmed in vivo that in mice injected with BxPC3 on their skin, there was significant reduction of tumor size in those treated with both F3.CE and BxPC3 adjacent to the cancer mass. Administration of F3.CE in conjunction with CPT-11 could be a new possibility as an effective treatment regimen for patients suffering from advanced pancreatic cancer.

Angiogenesis is a dynamic, multi-step process. It is known that about 70 diseases are related to angiogenesis. Both the experimental and the literature reports showed that Sophora flavescens inhibit angiogenesis significantly, but the material basis and the mechanism of action have not been clear. In this study, molecular docking was used for screening of anti-angiogenesis flavonoids from the roots of S. flavescens. One handred and twenty-six flavonoids selected from S. flavescens were screened in the docking ligand database with six targets(VEGF-a,TEK,KDR,Flt1,FGFR1 and FGFR2) as the receptors. In addition, the small-molecule approved drugs of targets from DrugBank database were set as a reference with minimum score of each target's approved drugs as threshold. The LibDock module in Discovery Studio 2.5 (DS2.5) software was applied to screen the compounds. As a result, 37 compounds were screened out that their scores were higher than the minimum score of approved drugs as well as being in the top of 10%. At last the mechanism of flavonoids anti-angiogenesis was preliminarily revealed, which provided a new method for the development of angiogenesis inhibitor drugs.

Vascular endothelial growth factor C (VEGFC) stimulates leukemia cell proliferation and survival, and promotes angiogenesis. We studied VEGFC expression in bone marrow samples from 353 adult acute myeloid leukemia (AML) patients and its relationship with several clinical, cytogenetic, and molecular variables. We also studied the expression of 84 genes involved in VEGF signaling in 24 patients. We found that VEGFC expression was higher in AML patients with myelodysplasia-related changes (AML-MRC) than in patients with non-AML-MRC. We also found an association between VEGFC expression and the patient cytogenetic risk group, with those with a worse prognosis having higher VEGFC expression levels. No correlation was observed between VEGFC expression and survival or complete remission. VEGFC expression strongly correlated with expression of the VEGF receptors FLT1, KDR, and NRP1. Thus, in this series, VEGFC expression was increased in AML-MRC and in subgroups with a poorer prognosis, but has no impact on survival.

Background: Neovascularization plays a critical role in skin graft survival. Up to date, the lack of specificity to solely track the newly sprouting blood vessels has remained a limiting factor in skin graft transplantation models. Therefore, the authors developed a new model by using Flt1-tdsRed BAC transgenic mice. Flt1 is a vascular endothelial growth factor receptor expressed by sprouting endothelial cells mediating neoangiogenesis. The authors determined whether this model reliably visualizes neovascularization by quantifying tdsRed fluorescence in the graft over 14 days.

Methods: Cross-transplantation of two full-thickness 1 × 1-cm dorsal skin grafts was performed between 6- to 8-week-old male Flt1 mice and KSN/Slc nude mice (n = 5). The percentage of graft area occupied by tdsRed fluorescence in the central and lateral areas of the graft on days 3, 5, 9, and 14 was determined using confocal-laser scanning microscopy.

Results: Flt1+ endothelial cells migrating from the transgenic wound bed into the nude graft were first visible in the reticular dermis of the graft center on day 3 (0.5 ± 0.1; p < 0.05). Peak neovascularization was observed on day 9 in the lateral and central parts, increasing by 2- to 4-fold (4.6 ± 0.8 and 4.2 ± 0.9; p < 0.001). Notably, some limited neoangiogenesis was displayed within the Flt grafts on nude mice, particularly in the center. No neovascularization was observed from the wound margins.

Conclusion: The ability of the Flt1-tdsRed transgenic mouse model to efficiently identify the origin of the skin-graft vasculature and visualize graft neovascularization over time suggests its potential utility for developing techniques that promote graft neovascularization.