BACKGROUND

Up-regulation of epithelial vascular endothelial growth factor (VEGF) expression was recently reported at an early stage of bladder carcinogenesis in rats. Subsequently, clotrimazole (CLT) was reported to exert an inhibitory effect on the in vitro proliferation of vascular endothelial cells stimulated by VEGF. The present study was performed to evaluate the effect of CLT on the initial stage of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced carcinogenesis of the urinary bladder in rats.

METHODS

Seven-week-old-female Wistar rats were divided into both a control group and the other four groups that were allowed free access to drinking water containing 0.05%BBN. Two groups of rats (10-CLT and 20-CLT) received CLT (100 mg/kg per day) once daily by gavage for 10 and 20 weeks, respectively. The other two groups of rats (10-BBN and 20-BBN) were maintained on the same BBN-containing drinking water for 10 and 20 weeks, respectively, without receiving CLT. After removing the urinary bladder of each rat, expression of VEGF and VEGF receptor FLT1, and new capillaries were examined by immunohistochemical staining. VEGF mRNA was detected by the reverse transcription polymerase chain reaction.

RESULTS

The ratio of VEGF-positive cells and the expression of VEGF mRNA were lower in the 20-CLT group than in the 10-BBN group. Newly formed capillaries in the 20-CLT group were significantly decreased compared with those in the 10-BBN group.

CONCLUSIONS

These quantitative data suggested that CLT can inhibit VEGF expression in vivo and hence inhibit angiogenesis in the early stages of bladder carcinoma.

Identifying rare variants that are responsible for complex disease has been promoted by advances in sequencing technologies. However, statistical methods that can handle the vast amount of data generated and that can interpret the complicated relationship between disease and these variants have lagged. We apply a zero-inflated Poisson regression model to take into account the excess of zeros caused by the extremely low frequency of the 24,487 exonic variants in the Genetic Analysis Workshop 17 data. We grouped the 697 subjects in the data set as Europeans, Asians, and Africans based on principal components analysis and found the total number of rare variants per gene for each individual. We then analyzed these collapsed variants based on the assumption that rare variants are enriched in a group of people affected by a disease compared to a group of unaffected people. We also tested the hypothesis with quantitative traits Q1, Q2, and Q4. Analyses performed on the combined 697 individuals and on each ethnic group yielded different results. For the combined population analysis, we found that UGT1A1, which was not part of the simulation model, was associated with disease liability and that FLT1, which was a causal locus in the simulation model, was associated with Q1. Of the causal loci in the simulation models, FLT1 and KDR were associated with Q1 and VNN1 was correlated with Q2. No significant genes were associated with Q4. These results show the feasibility and capability of our new statistical model to detect multiple rare variants influencing disease risk.

Existing methods for analyzing rare variant data focus on collapsing a group of rare variants into a single common variant; collapsing is based on an intuitive function of the rare variant genotype information, such as an indicator function or a weighted sum. It is more natural, however, to take into account the single-nucleotide polymorphism (SNP) interactions informed directly by the data. We propose a novel tree-based method that automatically detects SNP interactions and generates candidate markers from the original pool of rare variants. In addition, we utilize the advantage of having 200 phenotype replications in the Genetic Analysis Workshop 17 data to assess the candidate markers by means of repeated logistic regressions. This new approach shows potential in the rare variant analysis. We correctly identify the association between gene FLT1 and phenotype Affect, although there exist other false positives in our results. Our analyses are performed without knowledge of the underlying simulating model.

Multivariable analyses of global expression profiling are valid indicators of the prognosis of various diseases including brain cancers. To identify the candidates for markers of prognosis of glioblastoma, we performed multivariable analyses on the status of epithelial (EPI)-mesenchymal (MES) transition (EMT), glioma (GLI) stem cells (GSCs), molecular target therapy (MTT), and potential glioma biomarkers (PGBs) using the expression data and clinical information from patients. Random forest survival and Cox proportional hazards regression analyses indicated significant variable values for DSG3, CLDN1, CDH11, FN1, HDAC3/7, PTEN, L1CAM, OLIG2, TIMP4, IGFBP2, and GFAP. The analyses also comprised prognosis prediction formulae that could distinguish between the survival curves of the glioblastoma patients. In addition to the genes mentioned above, HDAC1, FLT1, EGFR, MGMT, PGF, STAT3, SIRT1, and GADD45A constituted complex genetic interaction networks. The calculated status scores obtained by principal component analysis indicated that GLI genes covered the status of EPI, GSC, and MTT-related genes. Moreover, survival tree analyses indicated that MES^high, MES^highGLI^low, GSC^highGLI^low, MES^highMTT^low, and PGB^high showed poor prognoses and MES^middle, GSC^low, and PGB^low showed good prognoses, suggesting that enhanced EMT and GSC are associated with poor survival and that lower expression of EPI markers and the pre-stages of EMT are relatively less malignant in glioblastoma. These results demonstrate that the assessment of EMT and GSC enables the prediction of the prognosis of glioblastoma that would help develop novel therapeutics and de novo marker candidates for the prognoses of glioblastoma.

We develop statistical methods for detecting rare variants that are associated with quantitative traits. We propose two strategies and their combination for this purpose: the iterative regression strategy and the extreme values strategy. In the iterative regression strategy, we use iterative regression on residuals and a multimarker association test to identify a group of significant variants. In the extreme values strategy, we use individuals with extreme trait values to select candidate genes and then test only these candidate genes. These two strategies are integrated into a hybrid approach through a weighting technology. We apply the proposed methods to analyze the Genetic Analysis Workshop 17 data set. The results show that the hybrid approach is the most powerful approach. Using the hybrid approach, the average power to detect causal genes for Q1 is about 40% and the powers to detect FLT1 and KDR are 100% and 68% for Q1, respectively. The powers to detect VNN3 and BCHE are 34% and 30% for Q2, respectively.

This paper introduces a differential network biology for discovering tumor migration. We applied statistical methods to prioritize PPI candidates and an in situ proximity ligation assay to verify 67 endogenous PPIs among 21 interlinked pathways in two hepatocellular carcinoma (HCC) cells, Huh7 (minimally migratory cells) and Mahlavu (highly migratory cells). Differential network biology analysis was applied to determine the novel interaction, CRKL-FLT1, has a high centrality ranking, and the expression of this interaction is strongly correlated with the migratory ability of HCC and other cancer cell lines. Knockdown of CRKL and FLT1 in HCC cells leads to a decrease in cell migration. This study demonstrated that functional exploration of a disease network with differential network in interlinked pathways via PPIs can be used to discover tumor migration.

BACKGROUND

Previous studies have revealed that women with gestational diabetes mellitus (GDM) have an increased risk of developing preeclampsia (PE). The possible reason is the abnormal lipid metabolism caused by GDM that leads to dysfunction of vascular endothelial cells and atherosclerosis, resulting in the onset of PE. However, studies focusing on the pathogenesis of PE in syncytiotrophoblast of GDM patients are lacking. This study aimed to compare differentially expressed proteins from syncytiotrophoblast between women with GDM and women with GDM with subsequently developed PE.

METHODS

Syncytiotrophoblast samples were obtained from pregnant women immediately after delivery. To explore the protein expression changes of syncytiotrophoblast that might explain the pathogenesis of PE in women with GDM, quantitative proteomics was performed using tandem mass tag (TMT) isobaric tags and liquid chromatography-tandem mass spectrometry. Bioinformatics analysis was performed to enrich the biological processes that these differentially expressed proteins were involved in.

RESULTS

A total of 28,234 unique peptides and 4140 proteins were identified in all samples. Among them, 23 differentially expressed proteins were identified between patients with GDM and patients with GDM with subsequently developed PE. Therein, 11 proteins were upregulated and 12 proteins were downregulated. Two relative proteins (FLT1 and PABPC4) were independently verified using immunoblotting analysis. Bioinformatic results indicated that the onset of PE in patients with GDM is a multifactorial disorder, involving factors such as apoptosis, transcriptional misregulation, oxidative stress, lipid metabolism, cell infiltration and migration, and angiogenesis.

CONCLUSIONS

These results indicated that the inadequacy of endometrium infiltration, angiogenic disorder, and oxidative stress in syncytiotrophoblast are more likely to occur in patients with GDM and may be the potential mechanisms leading to such patients secondarily developing severe early-onset PE.

OBJECTIVE

Anti-vascular endothelial growth factor (VEGF) antibody therapy is an effective treatment for ocular angiogenesis. Although the intraocular pressure of some patients increases after anti-VEGF therapy, the effects of VEGF-A on the aqueous humor outflow pathway remain unknown. This study investigated the effects of VEGF-A on the aqueous humor outflow pathway.

METHODS

We used human recombinant VEGF121 and VEGF165. Trabecular meshwork (TM) and Schlemm's canal endothelial (SCE) cells were isolated from the eyes of cynomolgus monkeys. Expression of mRNA coding four VEGF receptors, VEGFR1 (FLT1), VEGFR2 (KDR), neuropilin-1, and neuropilin-2, was examined by RT-PCR. To evaluate the permeability of cell monolayers, we measured transendothelial electrical resistance (TEER). The outflow facility was measured in perfused porcine anterior segment organ cultures treated with 30 ng/mL VEGF121 for 48 h.

RESULTS

Four VEGF-A-related receptor mRNAs were expressed in TM and SCE cells. The TEER of TM cells was not significantly affected by VEGF121 or VEGF165 treatment. In contrast, the TEER of SCE cells was significantly lower 48 h after treatment with 30 ng/mL VEGF121 to 69.4 ± 12.2% of baseline (n = 10), which was a significant difference compared with the control (P = 0.0001). VEGF165 (30 ng/mL) decreased the TEER of SCE cells at 48 h after treatment to 72.3 ± 14.1% compared with the baseline (n = 10), which was not a significant difference compared with the control (P = 0.0935). Ki8751, a selective VEGFR2 inhibitor, completely suppressed the effect of VEGF121 on SCE cell permeability, although ZM306416, a selective VEGFR1 inhibitor, did not affect the VEGF121-induced decrease in TEER. Perfusion with 30 ng/mL of VEGF121 for 48 h significantly increased the outflow facility compared with the control (47.8 ± 28.5%, n = 5, P = 0.013).

CONCLUSIONS

These results suggest that VEGF-A may regulate the conventional aqueous outflow of SCE cells through VEGFR2.

Lymphatic and blood vascular endothelial cells (ECs) share several molecular and developmental features. However, these two cell types possess distinct phenotypic signatures, reflecting their different biological functions. Despite significant advances in elucidating how the specification of lymphatic and blood vascular ECs is regulated at the transcriptional level during development, the key molecular mechanisms governing their lineage identity under physiological or pathological conditions remain poorly understood. To explore the epigenomic signatures in the maintenance of EC lineage specificity, we compared the transcriptomic landscapes, histone composition (H3K4me3 and H3K27me3) and DNA methylomes of cultured matched human primary dermal lymphatic and blood vascular ECs. Our findings reveal that blood vascular lineage genes manifest a more 'repressed' histone composition in lymphatic ECs, whereas DNA methylation at promoters is less linked to the differential transcriptomes of lymphatic versus blood vascular ECs. Meta-analyses identified two transcriptional regulators, BCL6 and MEF2C, which potentially govern endothelial lineage specificity. Notably, the blood vascular endothelial lineage markers CD34, ESAM and FLT1 and the lymphatic endothelial lineage markers PROX1, PDPN and FLT4 exhibited highly differential epigenetic profiles and responded in distinct manners to epigenetic drug treatments. The perturbation of histone and DNA methylation selectively promoted the expression of blood vascular endothelial markers in lymphatic endothelial cells, but not vice versa. Overall, our study reveals that the fine regulation of lymphatic and blood vascular endothelial transcriptomes is maintained via several epigenetic mechanisms, which are crucial to the maintenance of endothelial cell identity.

Keywords: Blood endothelial cells; Cell identity; DNA methylation; Epigenetics; Histone modifications; Lymphatic endothelial cells.

Biliary tract cancer (BTC) represents a heterogeneous disease with dismal outcome. Herein, we examined genotype and angiogenesis features in BTC. We applied genotyping (Sanger, qPCR, 101-gene panel NGS), mRNA relative quantification methods, and β-catenin immunohistochemistry in 84 FFPE BTC (55 gallbladder [GBC], 14 intrahepatic [ICC], 15 extrahepatic [ECC] carcinomas). We identified 541 mutations in 68 (81%) tumors. Top mutated genes were CTNNB1 (36%); PTEN (33%); TP53 (31%); PIK3R1 (29%); PIK3CA (13%); BRCA2 and KRAS (12%); BRCA1 (11%). Six GBCs were hypermutated [hm] displaying a distinct mutational pattern. Mutations in TP53 and PI3K, Wnt and RAS components were prevalent among non-hypermutated tumors. All hmGBCs carried mutations in BRCA2 and other homologous recombination repair (HRR) genes, in PD1, but not in CTNNB1 and KRAS. None of the pathogenic BRCA2 p.D2723G and BRCA1 p.Q563* and c.5266dupC was present at frequencies expected for germline mutations. We observed copy gains (>6 copies) in EGFR (9% of informative tumors), PRKAR1A (7%), PIK3CA (6%), ERBB2 (5%) and MET (4%). TP53 mutations were prevalent in GBC (P<0.001) and PRKAR1A copy gains in ICC (P=0.007). PTEN was frequently co-mutated with CTNNB1 (P<0.001). Unrelated to CTNNB1 mutations, nuclear β-catenin was detected in 45% of tumors, among them in 5/6 hmGBC. We observed strong mRNA expression correlation of the two neuropilins (NRP1 and NRP2) with each other (Spearman's rho 0.59) and with the endothelin receptor (NRP2 rho 0.66; NRP2 rho 0.51), and between VEGFA and its receptors (FLT1 rho 0.49; KDR rho 0.45). All PIK3CA mutated tumors expressed endothelin 1 mRNA (P=0.010). Most tumors expressing nuclear β-catenin were negative for VEGFC (P=0.009) and FLT4 (P=0.002) mRNA expression. In conclusion, we confirmed the presence of known genomic aberrations in BTC and different genotypes between BTC subsets. Novel findings are the coexistence of PI3K and WNT pathway gene alterations in BTC, their association with angiogenesis, and the hypermutated GBCs with HRR gene mutations, all of which may be considered for new treatment options in this difficult to treat disease.

OBJECTIVE

In order to search new therapeutic strategies, study the relationship between angiogenesis and vascular endothelial growth factor and its two receptors in lung carcinoma.

METHODS

The study consisted of 49 patients with lung carcinoma treated with curative surgery. Immunohistochemistry on paraffin sections was performed with anti-human factor VIII antibody to study the microvascular density (MVD), and with antibodies to VEGF, Flt1, and KDR to investigate the expression of these three proteins in different cellular compartments. The survival time was compared between low MVD and high MVD groups by the Kaplan-Meier method.

RESULTS

(1) Expression of VEGF and its two receptors in tumor cells, stromal fibroblasts and endothelial cells was demonstrated. (2) There was no significant difference between low MVD and high MVD groups in the clinical data, including TNM stage, clinical phase, pathologic type, and tumor cell differentiation (P>> 0.05). Survival analysis showed that the high MVD group was associated with a high risk of death. (3) There was no significant difference between low MVD and high MVD groups in VEGF expression in tumor cells, but in endothelial cells both Flt1 and KDR were correlated with high microvessel count. (4) The high expression of VEGF in tumor cells was correlated with that in stromal fibroblasts. The level of expression in both cells was consistent. (5) In both tumor cells and endothelial cells, high co-expression of VEGF and KDR was consistent, but that of VEGF and Flt1 showed inconsistency.

CONCLUSIONS

(1) Not or rarely affected by clinical factors, MVD is a good and independent prognostic factor for patients with lung carcinoma. (2) The angiogenesis is induced not only by VEGF itself, but via VEGF receptors too. VEGF and its receptors thus appear to be new therapeutic targets for lung carcinoma. (3) VEGF appears to be produced by both tumor cells and stromal fibroblasts. (4) The result suggests that tumor expansion and angiogenesis are mainly induced by an autocrine pathway and a paracrine loop of VEGF via KDR.

Approximately 90 % of head and neck cancers are squamous cell carcinomas (HNSCC), and the overall 5-year survival rate is not higher than 50 %. There is much evidence that human papillomavirus (HPV) infection may influence the expression of commonly studied HNSCC markers. Our study was focused on the possible HPV-specificity of molecular markers that could be key players in important steps of cancerogenesis (MKI67, EGF, EGFR, BCL-2, BAX, FOS, JUN, TP53, MT1A, MT2A, VEGFA, FLT1, MMP2, MMP9, and POU5F). qRT-PCR analysis of these selected genes was performed on 74 biopsy samples of tumors from patients with histologically verified HNSCC (22 HPV-, 52 HPV+). Kaplan-Meier analysis was done to determine the relevance of these selected markers for HNSCC prognosis. In conclusion, our study confirms the impact of HPV infection on commonly studied HNSCC markers MT2A, MMP9, FLT1, VEGFA, and POU5F that were more highly expressed in HPV-negative HNSCC patients and also shows the relevance of studied markers in HPV-positive and HPV-negative HNSCC patients.

Genetic and environmental factors have been shown to work together to alter cancer risk. In this study we evaluate previously identified gene and lifestyle interactions in a candidate pathway that were associated with colon cancer risk to see if these interactions altered gene expression. We analyzed non-tumor RNA-seq data from 144 colon cancer patients who had genotype, recent cigarette smoking, diet, body mass index (BMI), and recent aspirin/non-steroidal anti-inflammatory use data. Using a false discovery rate of 0.1, we evaluated differential gene expression between high and low levels of lifestyle exposure and genotypes using DESeq2. Thirteen pathway genes and 17 SNPs within those genes were associated with altered expression of other genes in the pathway. BMI, NSAIDs use and dietary components of the oxidative balance score (OBS) also were associated with altered gene expression. SNPs previously identified as interacting with these lifestyle factors, altered expression of pathway genes. NSAIDs interacted with 10 genes (15 SNPs) within those genes to alter expression of 28 pathway genes; recent cigarette smoking interacted with seven genes (nine SNPs) to alter expression of 27 genes. BMI interacted with FLT1, KDR, SEPN1, TERT, TXNRD2, and VEGFA to alter expression of eight genes. Three genes (five SNPs) interacted with OBS to alter expression of 12 genes. These data provide support for previously identified lifestyle and gene interactions associated with colon cancer in that they altered expression of key pathway genes. The need to consider lifestyle factors in conjunction with genetic factors is illustrated.

Activation of vascular endothelial growth factor receptor 1 (VEGFR1/FLT1) and 2 (VEGFR2/KDR) involves receptor dimerization. Formation of VEGFR dimer has so far not been visualized in single intact cells. In the present study we describe different optical assays which can be used to observe dimerization of VEGFR1 and VEGFR2. Bimolecular fluorescence complementation (BIFC) assays confirmed homo,- and heterodimerization of transfected receptors. Fluorescence resonance energy transfer (FRET) techniques in living and fixed CHO-K1 cells allowed observation of VEGFR1 homodimer,- and VEGFR1 and VEGFR2 heterodimer formation after ligand stimulation. After inhibition of ligand binding by the VEGFA JH121 antibody VEGFR1 homodimerization was completely abolished. Under the same conditions, cells transfected by VEGFR1 and VEGFR2 maintained relevant receptor heterodimerization. These techniques to monitor VEGFR1 and VEGFR2 homo- and heterodimerization in living and fixed cells may help in the search for new angiogenesis-directed inhibitors of VEGFR dimerization.

To enable the assessment of compound heterozygosity, we propose a simple approach for incorporating genotype phase in a rare variant collapsing procedure for the analysis of DNA sequence data. When multiple variants are identified within a gene, knowing the phase of each variant may provide additional statistical power to detect associations with phenotypes that follow a recessive or additive inheritance pattern. We begin by phasing all marker data; then, we collapse nonsynonymous single-nucleotide polymorphisms within genes on each phased haplotype, resulting in a single diploid genotype for each gene, which represents whether one or both haplotypes carry a nonsynonymous variant allele. A recessive or additive association test can then be used to assess the relationship between the collapsed genotype and the phenotype of interest. We apply this approach to the unrelated individuals data from Genetic Analysis Workshop 17 and compare the results of the additive test with a dominant test in which phase is not informative. Analysis of the first phenotype replicate shows that the FLT1 gene is significantly associated with both Q1 and the binary affection status phenotype. This association was detected by both the additive and dominant tests, although the additive phase-informed test resulted in a smaller p-value. No false-positive results were detected in the first phenotype replicate. Analysis of the average values of all phenotype replicates correctly identified five other genes important to the simulation, but with an increase in false-positive rates. The accuracy of our method is contingent on correct phase determination.

Vascular endothelial growth factor (VEGF) is a key player in vasculogenesis and angiogenesis in the embryo, and essential in neovascularization in adults. Natural VEGF inhibitors such as soluble VEGF receptors, among them the soluble VEGF-trapping receptor Flt1 (sFlt1), participate in VEGF regulation. Decreased levels of VEGF and increased levels of sFlt1 have been implicated in the pathophysiology of preeclampsia. We discovered a soluble receptor, sFlt1-14, qualitatively different from sFlt1 and a potent VEGF inhibitor. It is generated in a cell type specific fashion, primarily in nonendothelial cells, most notably in vascular smooth muscle cells. We showed that increased production of soluble VEGF receptors in pregnancy is owing to expression of sFlt1-14, from the end of the first trimester to term. This expression is markedly elevated in preeclampsia, and is expressed chiefly by syncitial knots. In subsequent studies we found that sFlt1 is a strong heparin binder: this capability enables it to stay attached to blood vessels and to the placenta. Ex vivo, sFlt1 can be heparin displaced to medium from aortic segments and placental villi. In vivo, pregnant women treated with the low molecular weight heparin (LMWH) have elevated sFlt1 levels in their circulations. Interestingly, LMWH raised VEGF levels over and above the increase in sFlt1 levels in these patients. Heparanaseoverexpressing non-pregnant as well as pregnant transgenic mice present elevated levels of sFlt1 in their circulations. Ex vivo prevention of heparanase maturation through cathepsin L inhibition, or targeting heparanase directly with a neutralizing antibody, both resulted in a marked reduction in sFlt1 secretion to medium of normal and preeclamptic placental expiants. These findings uncover a new level of regulation that controls sFlt1 bio-distribution, and directs it to function in the vicinity of its producing cell. Heparanase or LMWH has the ability to liberate sFlt1 from its retention, so this process may be a potential target for preeclampsia treatment.

BACKGROUND

Osteoarthritis (OA) is the most common chronic joint disease. This study aimed to uncover underlying mechanisms of OA pathogenesis and explore the potential biomarkers of osteoarthritic acetabular labrum.

METHODS

The microarray data GSE60762 was utilized, containing five OA acetabular labrum samples and three healthy control samples. Data were preprocessed by oligo package and the differentially expressed genes (DEGs) were identified using limma package with predefined criteria, followed by functional enrichment analysis by the GoFunction in R Bioconductor, and protein-protein interaction (PPI) network analysis.

RESULTS

As a result, 141 DEGs (44 were up-regulated and 97 were down-regulated) were identified between OA and healthy acetabular labrum cells. Up-regulated genes including CDH2 and WNT5A were significantly enriched in intracellular signal transduction function, while down-regulated genes such as KDR, FLT1 and CDH5 were remarkably correlated with cardiovascular system development. FLT1, KDR, CDH2 and CDH5 were the striking nodes in the PPI network.

CONCLUSIONS

CDH2, WNT5A, KDR, FLT1 and CDH5 might serve as the biomarkers of OA prognosis. Intracellular signal transduction and cardiovascular system development might play significant roles in the destruction of labrum during OA progression. However, more experimental validations are warranted to confirm our findings.

Anti-angiogenic therapy represents a promising, new therapeutic modality for malignant gliomas. The present study was designed to define the malignant glioma cases most suitable for anti-angiogenic therapy and to demonstrate the efficacy of anti-angiogenic therapy using soluble-form Flt1 (sFlt1) gene delivery in mice. In human malignant glioma samples (39 glioblastomas, 21 anaplastic astrocytomas and 4 anaplastic oligoastrocytomas), protein expression of VEGF, and its specific natural inhibitor, sFlt1, as well as vessel architecture were assessed. Among these variables, VEGF >1000 ng/ml, VEGF/sFlt1 ratio >1, vessel density >30, and vessel area >7% were prognostic factors for malignant gliomas. VEGF/sFlt1 ratio >1 was the most powerful prognostic marker for survival in multivariate analysis. The sFlt1 gene was also successfully introduced into U87 glioma cells in vitro, resulting in 31% tumor growth inhibition in vivo. sFlt1-transfected tumor demonstrated high sFlt-1 expression along with diminished vessel density and area compared with the control tumor. In transfected tumor, VEGF expression was decreased in the viable area, but still high in the hypoxic area. sFlt1 and VEGF expression was re-evaluated in vitro using glioma cells under normoxic and hypoxic conditions. For sFlt1-transfected cells, VEGF expression was upregulated, but sFlt1 expression was downregulated, resulting in an increase of VEGF/sFlt1 ratio in hypoxic conditions. We conclude that malignant gliomas with a high VEGF/sFlt1 ratio and large vessel area are good candidates for anti-angiogenic therapy. Soluble Flt1 gene delivery was demonstrated to inhibit glioma growth, but this was limited in hypoxic areas.

Previous mapping between the human and pig genomes suggested extensive conservation of human chromosome 13 (HSA13) to pig chromosome 11 (SSC11). The objectives of this study were comparative gene mapping of pig homologs of HSA13 genes and examining gene order within this conserved synteny group by physical assignment of each locus. A detailed HSA13 to SSC11 comparison was chosen since the comparative gene map is not well developed for these chromosomes and a rearranged gene order within conserved synteny groups was observed from the comparison between HSA13 and bovine chromosome 12 (BTA12). Heterologous primers for PCR were designed and used to amplify pig homologous fragments. The pig fragments were sequenced to confirm the homology. Six pig STSs (FLT1, ESD, RB1, HTR2A, EDNRB, and F10) were physically mapped using a somatic cell hybrid panel to SSC11, and fluorescent in situ hybridization (FISH) mapping was also applied to improve map resolution and determine gene order. Results from this study increase the comparative information available on SSC11 and suggest a conserved gene order on SSC11 and HSA13, in contrast to human:bovine comparisons of this syntenic group.

The soluble vascular endothelial growth factor (VEGF) receptor 1 (sFLT1) has been tested in both animals and humans for anti-angiogenic therapies, for example, age-related macular degeneration. We hypothesized that adeno-associated viral vector (AAV)-mediated sFLT1 expression could be used to inhibit abnormal brain angiogenesis. We tested the anti-angiogenic effect of sFLT1 and the feasibility of using AAV serotype 9 to deliver sFLT1 through intravenous injection (IV) to the brain angiogenic region. AAVs were packaged in AAV serotypes 1 and 2 (stereotactic injection) and 9 (IV injection). Brain angiogenesis was induced in adult mice through stereotactic injection of AAV1-VEGF. AAV2-sFLT02 containing sFLT1 VEGF-binding domain (domain 2) was injected into the brain angiogenic region, and AAV9-sFLT1 was injected into the jugular vein at the time of or 4 weeks after AAV1-VEGF injection. We showed that AAV2-sFLT02 inhibited brain angiogenesis at both time points. IV injection of AAV9-sFLT1 inhibited angiogenesis only when the vector was injected 4 weeks after angiogenic induction. Neither lymphocyte infiltration nor neuron loss was observed in AAV9-sFLT1-treated mice. Our data show that systemically delivered AAV9-sFLT1 inhibits angiogenesis in the mouse brain, which could be utilized to treat brain angiogenic diseases such as brain arteriovenous malformation.

In this paper, we propose an intravitreal implantable magnetic micropump integrated with micro check valve capable of on-demand vascular endothelial growth factor receptor (VEGFR)-targeted drug delivery for the treatment of age-related macular degeneration, diabetic retinopathy and other eye pathologies characterized by ocular neoangiogenesis. Precise on-demand drug release is realized by the deflection of the magnetic membrane assembly according to the external magnetic field, and the membrane assembly consists of a thin elastic polydimethylsiloxane (PDMS) membrane and a cylindrical magnetic nanoparticle-PDMS composite block. Additionally, a micro check valve composed of two PDMS layers was integrated into the micropump to realize a diode-like one-directional drug delivery and prevent undesired drug diffusion. For specifically targeting VEGFR and suppression of VEGF-induced proliferation of microvascular endothelial cells, anti-Flt1 gold nanocomplexes are synthesized. In vitro and in vivo experiments and quantitative analysis are carried out in order to verify our proposed concept: precise drug release control according to the external magnetic field, targeting to microvascular endothelial cells, and efficient and on-demand drug delivery from the proposed micropump to the macular area of rabbit's eye.

We have previously reported that the major isoform of Flt1/VEGFR-1 expressed in MDA-MB-231 breast cancer cells was a truncated intracellular isoform transcribed from intron 21 (i21 Flt1). This isoform upregulated the active form of Src and increased breast cancer cell invasiveness. Since expression of the transmembrane and soluble Flt1 isoforms of HUVEC is activated by Notch signaling, we wondered whether the expression of the intracellular isoform i21 Flt1 was also dependent on Notch activation. We report here that the expression of i21 Flt1 in HUVEC and MDA-MB-231 cells is downregulated by the γ-secretase inhibitor DAPT. In addition, treatment of MDA-MB-231 cells with siRNA specific for Notch-1 and Notch-3 downregulates the expression of i21 Flt1. In agreement with these findings, HUVEC and MDA-MB-231 breast cancer cells, cultured on dishes coated with recombinant human Dll4 extracellular domain, express higher levels of i21 Flt1. In cancer cells, Flt1 is a target of the micro RNA family miR-200. In MDA-MB-231 breast cancer cells, the truncated intracellular isoform i21 Flt1 is also negatively regulated by miR-200c. Retinoic acid interferes i21 Flt1 expression by downregulating Notch-3 and upregulating miR-200 expression. Treatment of MDA-MB-231 breast cancer cells with both a γ-secretase inhibitor and retinoic acid suppresses the expression of i21 Flt1, providing a new mechanism to explain the effectiveness of this therapeutic approach.