• VEGFR
• VEGFxxxa
• VEGFxxxb
• alternative splicing
• angiogenesis
• cancer
• full-length VEGF

Endothelial cell (EC) activity is essential for tissue regeneration in several (patho)physiological contexts. However, our capacity to deliver in vivo biomolecules capable of controlling EC fate is relatively limited. Here, we screened a library of microRNA (miR) mimics and identified 25 miRs capable of enhancing the survival of ECs exposed to ischemia-mimicking conditions. In vitro, we showed that miR-425-5p, one of the hits, was able to enhance EC survival and migration. In vivo, using a mouse Matrigel plug assay, we showed that ECs transfected with miR-425-5p displayed enhanced survival compared with scramble-transfected ECs. Mechanistically, we showed that miR-425-5p modulated the PTEN/PI3K/AKT pathway and inhibition of miR-425-5p target genes (DACH1, PTEN, RGS5, and VASH1) phenocopied the pro-survival. For the in vivo delivery of miR-425-5p, we modulated small extracellular vesicles (sEVs) with miR-425-5p and showed, in vitro, that miR-425-5p-modulated sEVs were (1) capable of enhancing the survival of ECs exposed to ischemia-mimic conditions, and (2) efficiently internalized by skin cells. Finally, using a streptozotocin-induced diabetic wound healing mouse model, we showed that, compared with miR-scrambled-modulated sEVs, topical administration of miR-425-5p-modulated sEVs significantly enhanced wound healing, a process mediated by enhanced vascularization and skin re-epithelialization.

• angiogenesis
• endothelial cells
• extracellular vesicles
• high-throughput screening
• microRNAs
• non-coding RNAs
• pro-survival
• wound healing

• Endothelial cells
• Hepatocellular carcinoma
• Metastasis
• Tumor protein 53 (TP53)
• β-Catenin

• Head and neck squamous cell carcinoma
• MTDH
• Macrophages
• VEGFA-165/Flt-1 axis
• Vasculogenic mimicry

• Animals
• Antigens, CD/metabolism
• Cadherins/metabolism
• Cell Line, Tumor
• Cell Movement
• Female
• Head and Neck Neoplasms/metabolism
• Head and Neck Neoplasms/pathology
• Humans
• Macrophage Activation
• Macrophages/metabolism
• Male
• Membrane Proteins/metabolism
• Mice, Inbred BALB C
• Mice, Nude
• Middle Aged
• Neoplasm Metastasis
• Neovascularization, Pathologic/metabolism
• Nuclear Proteins/metabolism
• RNA-Binding Proteins/metabolism
• STAT3 Transcription Factor/metabolism
• Signal Transduction
• Squamous Cell Carcinoma of Head and Neck/metabolism
• Squamous Cell Carcinoma of Head and Neck/pathology
• Tumor-Associated Macrophages/metabolism
• Twist-Related Protein 1/metabolism
• Vascular Endothelial Growth Factor A/metabolism
• Vascular Endothelial Growth Factor Receptor-1/metabolism
• beta Catenin/metabolism
• Mice

• G-quadruplex
• aptamer
• design
• dimerization
• molecular recognition
• multivalency
• protein target
• therapy

• Anti-Inflammatory Agents/chemistry
• Anticoagulants/chemistry
• Antineoplastic Agents/chemistry
• Antiviral Agents/chemistry
• Aptamers, Nucleotide/chemistry
• CD3 Complex/chemistry
• Cell Adhesion Molecules/chemistry
• DNA/chemistry
• Dimerization
• G-Quadruplexes
• Humans
• Immunoglobulin M/chemistry
• Phosphoproteins/chemistry
• Protein Structure, Secondary
• Proto-Oncogene Proteins c-met/chemistry
• Pyrrolidines/chemistry
• RNA-Binding Proteins/chemistry
• Receptor Protein-Tyrosine Kinases/chemistry
• Receptors, Antigen, T-Cell/chemistry
• Vascular Endothelial Growth Factor A/metabolism
• Vitronectin/chemistry
• Nucleolin

• DNA-based aptamers
• G-quadruplex
• VEGF
• anticancer agents
• diagnostics
• therapy

Alternative splicing (AS) provides the primary mechanism for producing protein diversity. There is growing evidence that AS is involved in the development and progression of cancers. The rapid accumulation of high‐throughput sequencing technologies and clinical data sets offers an opportunity to systematically profile the relationship between mRNA variants and clinical outcomes. However, there is a lack of systematic analysis of AS in prostate cancer: Download RNA‐seq data and clinical information from The Cancer Genome Atlas (TCGA) data portal. Evaluate RNA splicing patterns by SpliceSeq and calculate splicing percentage (PSI) values. Different expressions were identified as differently expressed AS events (DEAs) based on PSI values. Bioinformatics methods were used for further analysis of DEAs and their splicing networks. Kaplan‐Meier, Cox proportional regression, and unsupervised cluster analysis were used to assess the correlation between DEAs and clinical characteristics. In total, 43 834 AS events were identified, of which 1628 AS events were differentially expressed. The parental genes of these DEAs played a significant role in the regulation of prostate cancer‐related processes. In total, 226 DEAs events were found to be associated with disease‐free survival. Four clusters of molecules with different survival modes were revealed by unsupervised cluster analysis of DEAs. AS events may be important determinants of prognosis and bio‐modulation in prostate cancer. In this study, we established strong prognostic predictors, discovered a splicing network that may be a potential mechanism, and provided further validated therapeutic targets.

• RNA-seq
• alternative splicing
• prognosis
• prostate cancer

• G-quadruplexes
• V7t1
• VEGF165
• aptamers
• biophysical characterization
• covalent dimers

• Aptamers, Nucleotide/chemistry
• Aptamers, Nucleotide/pharmacology
• Cell Proliferation/drug effects
• G-Quadruplexes
• Humans
• MCF-7 Cells
• Protein Binding
• Vascular Endothelial Growth Factor A/metabolism

• Myc
• PEGASAS
• alternative splicing
• prostate cancer
• rMATS

The G-quadruplex-forming VEGF-binding aptamer V7t1 was previously found to be highly polymorphic in a K⁺-containing solution and, to restrict its conformational preferences to a unique, well-defined form, modified nucleotides (LNA and/or UNA) were inserted in its sequence. We here report an in-depth biophysical characterization of V7t1 in a Na⁺-rich medium, mimicking the extracellular environment in which VEGF targeting should occur, carried out combining several techniques to analyse the conformational behaviour of the aptamer and its binding to the protein. Our results demonstrate that, in the presence of high Na⁺ concentrations, V7t1 behaves in a very different way if subjected or not to annealing procedures, as evidenced by native gel electrophoresis, size exclusion chromatography and dynamic light scattering analysis. Indeed, not-annealed V7t1 forms both monomeric and dimeric G-quadruplexes, while the annealed oligonucleotide is a monomeric species. Remarkably, only the dimeric aptamer efficiently binds VEGF, showing higher affinity for the protein compared to the monomeric species. These findings provide new precious information for the development of improved V7t1 analogues, allowing more efficient binding to the cancer-related protein and the design of effective biosensors or theranostic devices based on VEGF targeting.

• Angiogenesis Inhibitors/administration & dosage
• Animals
• Bevacizumab/administration & dosage
• Combined Modality Therapy/methods
• Drug Delivery Systems
• Female
• Humans
• Male
• Neoplasms/blood supply
• Neoplasms/therapy
• Neovascularization, Pathologic/prevention & control
• Prognosis
• Radiotherapy/methods
• Treatment Outcome
• Tumor Microenvironment/drug effects
• Tumor Microenvironment/radiation effects
• Vascular Endothelial Growth Factor A/drug effects
• Vascular Endothelial Growth Factor A/metabolism

Over 95% of human genes are alternatively spliced, expressing splice isoforms that often exhibit antagonistic functions. We describe genes whose alternative splicing has been linked to prostate cancer; namely VEGFA, KLF6, BCL2L2, ERG, and AR. We discuss opportunities to develop novel therapies that target specific splice isoforms, or that target the machinery of splicing. Therapeutic approaches include the development of small molecule inhibitors of splice factor kinases, splice isoform specific siRNAs, and splice switching oligonucleotides.

• AR
• BCL2L2
• ERG
• KLF6
• RNA interference
• VEGFA
• alternative splicing
• prostate cancer
• splice factor kinases
• splice factors
• splice switching oligonucleotides

• Alternative Splicing
• Apoptosis Regulatory Proteins/genetics
• Apoptosis Regulatory Proteins/metabolism
• Genetic Therapy/methods
• Humans
• Kruppel-Like Factor 6/genetics
• Kruppel-Like Factor 6/metabolism
• Male
• Prostatic Neoplasms/genetics
• Prostatic Neoplasms/therapy
• Receptors, Androgen/genetics
• Receptors, Androgen/metabolism
• Transcriptional Regulator ERG/genetics
• Transcriptional Regulator ERG/metabolism
• Vascular Endothelial Growth Factor A/genetics
• Vascular Endothelial Growth Factor A/metabolism

While we previously demonstrated that CITED2 expression in primary breast tumor tissues is elevated relative to normal mammary epithelium and inversely correlated with patient survival, its functional impact on primary tumor development and progression remained unknown. To address this issue, we examined the effect of CITED2 silencing on the growth of human breast cancer cell lines MDA-MB-231 and MDA-MB-468 following orthotopic administration in vivo. Here, we show that CITED2 silencing significantly attenuated MDA-MB-231 primary tumor growth concordant with reduced tumor vascularization, while MDA-MB-468 primary tumor growth and tumor vascularization remained unaffected. Correspondingly, expression of VEGFA was significantly reduced in shCITED2-expressing MDA-MB-231, but not MDA-MB-468 tumors. Consistent with the observed pattern of vascularization and VEGFA expression, we found that TGF-β stimulation induced expression of VEGFA and enhanced CITED2 recruitment to the VEGFA promoter in MDA-MA-231 cells, while failing to induce VEGFA expression in MDA-MB-468 cells. Further supporting its involvement in TGF-β-induced expression of VEGFA, CITED2 silencing prevented TGF-β induction of VEGFA expression in MDA-MB-231 cells. Collectively, these data indicate that CITED2 regulates primary breast tumor growth, likely by influencing tumor vasculature via TGF-β-dependent regulation of VEGFA.

• CITED2
• TGF-β
• VEGFA isoforms
• primary tumor growth
• vasculature

Several cell-based therapies are under pre-clinical and clinical evaluation for the treatment of ischemic diseases. Poor survival and vascular engraftment rates of transplanted cells force them to work mainly via time-limited paracrine actions. Although several approaches, including the use of soluble vascular endothelial growth factor (sVEGF)—VEGF₁₆₅, have been developed in the last 10 years to enhance cell survival, they showed limited efficacy. Here, we report a pro-survival approach based on VEGF-immobilized microparticles (VEGF-MPs). VEGF-MPs prolong VEGFR-2 and Akt phosphorylation in cord blood-derived late outgrowth endothelial progenitor cells (OEPCs). In vivo, OEPC aggregates containing VEGF-MPs show higher survival than those treated with sVEGF. Additionally, VEGF-MPs decrease miR-17 expression in OEPCs, thus increasing the expression of its target genes CDKN1A and ZNF652. The therapeutic effect of OEPCs is improved in vivo by inhibiting miR-17. Overall, our data show an experimental approach to improve therapeutic efficacy of proangiogenic cells for the treatment of ischemic diseases.

Soluble vascular endothelial growth factor (VEGF) enhances vascular engraftment of transplanted cells but the efficacy is low. Here, the authors show that VEGF-immobilized microparticles prolong survival of endothelial progenitors in vitro and in vivo by downregulating miR17 and upregulating CDKN1A and ZNF652.

Previous studies have suggested that microRNA-107 (miR-107) regulates cell migration in tumor and promotes Hypoxia Inducible Factor 1α (HIF1α) regulated angiogenesis under hypoxia. We found that miR-107 was strongly expressed in ischemic boundary zone (IBZ) after permanent middle cerebral artery occlusion (pMCAO) in rats and inhibition of miR-107 could reduce capillary density in the IBZ after stroke. Such finding led us to hypothesize that miR-107 might regulate post-stroke angiogenesis and therefore serve as a therapeutic target. We also found that antagomir-107, a synthetic miR-107 inhibitor, decreased the number of capillaries in IBZ and increased overall infarct volume after pMCAO in rats. We demonstrated that miR-107 could directly down-regulate Dicer-1, a gene that encodes an enzyme essential for processing microRNA (miRNA) precursors. This resulted in translational desupression of VEGF (vascular endothelial growth factor) mRNA, thereby increasing expression of endothelial cell-derived VEGF (VEGF165/VEGF164), leading to angiogenesis after stroke. This process might be a protective mechanism for ischemia-induced cerebral injury and miR-107 might be used as a novel tool in stroke treatment.

• Alternative splicing
• Amyloid beta precursor protein
• Colorectal cancer
• Numb homolog
• Vascular endothelial growth factor A

• Adenocarcinoma/genetics
• Adenocarcinoma/pathology
• Alternative Splicing
• Amplified Fragment Length Polymorphism Analysis
• Amyloid beta-Protein Precursor/genetics
• Colorectal Neoplasms/genetics
• Colorectal Neoplasms/pathology
• Female
• Gene Expression Regulation, Neoplastic
• Humans
• Male
• Membrane Proteins/genetics
• Nerve Tissue Proteins/genetics
• Polymorphism, Restriction Fragment Length
• RNA, Messenger/genetics
• Real-Time Polymerase Chain Reaction
• Reverse Transcriptase Polymerase Chain Reaction
• Vascular Endothelial Growth Factor A/genetics

• RNA-binding proteins
• alternative splicing
• breast cancer
• splicing factors

• alternative splicing
• splicing factor
• cancer
• metastasis
• rna-seq

• Alternative Splicing
• Animals
• Cell Line, Tumor
• Gene Expression Profiling/methods
• Gene Expression Regulation, Neoplastic
• Humans
• Male
• Mice
• Models, Biological
• Molecular Sequence Data
• Neoplasm Metastasis
• Oligonucleotide Array Sequence Analysis/methods
• Prostatic Neoplasms/genetics
• Prostatic Neoplasms/pathology
• RNA, Messenger/genetics
• Sequence Analysis, RNA

• P50 CA097186 NCI NIH HHS
• R01 GM105431 NIGMS NIH HHS
• R01 CA130916 NCI NIH HHS
• CA130916 NCI NIH HHS
• GM105431 NIGMS NIH HHS

Mesenchymal stem cells (MSCs) are multipotent and have been derived from various tissues. Although MSCs share many basic features, they often display subtle tissue specific differences. We previously demonstrated that bone marrow (BM) MSCs frequently become polyploid in culture. This tendency was mediated by a reduction in the expression of H19 long non-coding RNA during the transition from a diploid to a polyploid state.

MSCs were derived from both BM and adipose tissue of mice and expanded under normoxic and hypoxic culture conditions. Cells were stained by propidium iodide and their ploidy was evaluated by FACS. Gene expression of independent MSC preparations was compared by quantitative real time PCR and protein expression levels by Western blot analysis. p53 silencing in MSCs was performed by a specific small hairpin RNA (shRNA).

We set to examine whether genomic instability is common to MSCs originating from different tissues. It is demonstrated that adipose derived MSCs (ASCs) tend to remain diploid during culture while a vast majority of BM MSCs become polyploid. The diploid phenotype of ASCs is correlated with reduced H19 expression compared to BM MSCs. Under hypoxic conditions (3% oxygen) both ASCs and BM MSCs demonstrate increased RNA expression of H19 and Vascular endothelial growth factor A. Importantly, ASC gene expression is significantly less variable than BM MSCs under both oxygen conditions, indicating to their superior homogeneity. Gene expression analysis revealed that p53 target genes, often induced by DNA damage, are up-regulated in ASCs under basal conditions. However, p53 activation following treatment with DNA damaging agents was strongly elevated in BM MSCs compared to ASCs. We found that p53 is involved in maintaining the stable diploid state of ASCs as p53 shRNA induced ploidy changes in ASCs but not in BM MSCs.

The increased genomic stability of murine ASCs together with their lower H19 expression and relative homogeneity suggest a tissue specific higher stability of ASCs compared to BM MSCs, possibly due to higher activity of p53. The tissue specific differences between MSCs from a different tissue source may have important consequences on the use of various MSCs both in vitro and in vivo.

The online version of this article (doi:10.1186/scrt529) contains supplementary material, which is available to authorized users.

• Angiogenesis
• Endothelial tube formation assay
• VEGF121

Chronic inflammation leads to the formation of a pro-tumorigenic microenvironment that can promote tumor development, growth and differentiation through augmentation of tumor angiogenesis. Prostate cancer (CaP) risk and prognosis are adversely correlated with a number of inflammatory and angiogenic mediators, including Toll-like receptors (TLRs), NF-κB and vascular endothelial growth factor (VEGF). Peroxiredoxin 1 (Prx1) was recently identified as an endogenous ligand for TLR4 that is secreted from CaP cells and promotes inflammation. Inhibition of Prx1 by CaP cells resulted in reduced expression of VEGF, diminished tumor vasculature and retarded tumor growth. The mechanism by which Prx1 regulates VEGF expression in normoxic conditions was investigated in the current study. Our results show that incubation of mouse vascular endothelial cells with recombinant Prx1 caused increases in VEGF expression that was dependent upon TLR4 and required hypoxia inducible factor-1 (HIF-1) interaction with the VEGF promoter. The induction of VEGF was also dependent upon NF-κB; however, NF-κB interaction with the VEGF promoter was not required for Prx1 induction of VEGF suggesting that NF-κB was acting indirectly to induce VEGF expression. The results presented here show that Prx1 stimulation increased NF-κB interaction with the HIF-1α promoter, leading to enhanced promoter activity and increases in HIF-1α mRNA levels, as well as augmented HIF-1 activity that resulted in VEGF expression. Prx1 induced HIF-1 also promoted NF-κB activity, suggesting the presence of a positive feedback loop that has the potential to perpetuate Prx1 induction of angiogenesis. Strikingly, inhibition of Prx1 expression in CaP was accompanied with reduced expression of HIF-1α. The combined findings of the current study and our previous study suggest that Prx1 interaction with TLR4 promotes CaP growth potentially through chronic activation of tumor angiogenesis.

• fatty acid synthase
• melanoma
• metastasis
• angiogenesis
• vascular endothelial growth factor a (vegfa)
• orlistat

• Animals
• Antineoplastic Agents/pharmacology
• Apoptosis/drug effects
• Carcinoma, Squamous Cell/drug therapy
• Cell Proliferation/drug effects
• Cell Survival/drug effects
• Enzyme Inhibitors/pharmacology
• Enzyme-Linked Immunosorbent Assay
• Fatty Acid Synthases/antagonists & inhibitors
• Gene Expression Regulation, Neoplastic/drug effects
• Humans
• Lactones/pharmacology
• Lung Neoplasms/prevention & control
• Lung Neoplasms/secondary
• Melanoma/blood supply
• Melanoma/drug therapy
• Melanoma/secondary
• Melanoma, Experimental/drug therapy
• Melanoma, Experimental/pathology
• Mice
• Mice, Inbred C57BL
• Mouth Neoplasms/drug therapy
• Neovascularization, Pathologic/prevention & control
• Orlistat
• RNA, Small Interfering
• Reverse Transcriptase Polymerase Chain Reaction
• Vascular Endothelial Growth Factor A/drug effects
• Vascular Endothelial Growth Factor A/metabolism

• vegf
• splicing
• angiogenesis

• Angiogenic Proteins/physiology
• Humans
• Protein Isoforms/physiology
• Vascular Endothelial Growth Factor A/genetics
• Vascular Endothelial Growth Factor A/physiology
• Vascular Endothelial Growth Factor B/genetics
• Vascular Endothelial Growth Factor B/physiology
• Vascular Endothelial Growth Factors/genetics
• Vascular Endothelial Growth Factors/physiology

• 058083 Wellcome Trust
• 79633 Wellcome Trust
• BS06/005 British Heart Foundation
• BS/06/005/20340 British Heart Foundation
• Wellcome Trust
• 079663 Wellcome Trust

Vascular endothelial growth factor-A (VEGF-A) has several isoforms, which differ in their capacity to bind extracellular matrix proteins and also in their affinity for VEGF receptors. Although the relative contribution of the VEGF isoforms has been studied in tumor angiogenesis, little is known about the mechanisms that regulate the alternative splicing process. Here, we tested microenvironment cues that might regulate VEGF alternative splicing. To test this, we used endometrial cancer cells that produce all VEGF isoforms as a model, and exposed them to varying pH levels, hormones, glucose and CoCl₂ (to mimic hypoxia). Low pH had the most consistent effects in inducing variations in VEGF splicing pattern (VEGF121 increased significantly, p < 0.001, when compared to VEGF145, 165 or 189). This was accompanied by activation of the p38 stress pathway and SR proteins (splicing factors) expression and phosphorylation. SF2/ASF, SRp20 and SRp40 down-regulation by siRNA impaired the effects of pH stimulation, blocking the shift in VEGF isoforms production. Taken together, we show for the first time that acidosis (low pH) regulates VEGF-A alternative splicing, may be through p38 activation and suggest the possible SR proteins involved in this process.

Vascular endothelial growth factor A (VEGF-A) is a potent secreted mitogen critical for physiological and pathological angiogenesis. Regulation of VEGF-A occurs at multiple levels, including transcription, mRNA stabilization, splicing, translation and differential cellular localization of various isoforms. Recent advances in our understanding of the posttranscriptional regulation of VEGF-A are comprised of the identification of stabilizing mRNA-binding proteins and the discovery of two internal ribosomal entry sites (IRES) as well as two alternative initiation codons in the 5′UTR of the VEGF-A mRNA. We have previously reported that VEGF-A translation initiation at both the AUG and CUG codons is dependent on the exon content of the coding region. In this report, we show that the expression of different VEGF-A isoforms is regulated by a small upstream open reading frame (uORF) located within an internal ribosome entry site, which is translated through a cap-independent mechanism. This uORF acts as a cis-regulatory element that regulates negatively the expression of the VEGF 121 isoform. Our data provide a framework for understanding how VEGF-A mRNAs are translated, and how the production of the VEGF 121 isoform is secured under non-hypoxic environmental conditions.