A novel multiplexed immunoassay identifies CEA, IL-8 and prolactin as prospective markers for Dukes' stages A-D colorectal cancers.

Background: Current methods widely deployed for colorectal cancers (CRC) screening lack the necessary sensitivity and specificity required for population-based early disease detection. Cancer-specific protein biomarkers are thought to be produced either by the tumor itself or other tissues in response to the presence of cancers or associated conditions. Equally, known examples of cancer protein biomarkers (e.

WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing.

Angiogenesis is regulated by the balance of proangiogenic VEGF(165) and antiangiogenic VEGF(165)b splice isoforms. Mutations in WT1, the Wilms' tumor suppressor gene, suppress VEGF(165)b and cause abnormal gonadogenesis, renal failure, and Wilms' tumors. In WT1 mutant cells, reduced VEGF(165)b was due to lack of WT1-mediated transcriptional repression of the splicing-factor kinase SRPK1.

A human neutralizing antibody specific to Ang-2 inhibits ocular angiogenesis.

Objective: Angiogenesis, a critical contributor to ocular as well as neoplastic diseases, is stimulated by endothelial production of angiopoietin-2 (Ang2). Our objective was to determine the requirement of ocular angiogenesis for Ang2 in animal models of disease.

Methods: We developed and compared the effect of a novel human Ang2 antibody with a pan-angiopoietin strategy on angiogenesis in ocular angiogenesis in animal models of oxygen-induced retinopathy, and laser photocoagulation and confirmed its efficacy in xenografted human colorectal tumors.

Antiangiogenic isoforms of vascular endothelial growth factor predominate in subretinal fluid of patients with rhegmatogenous retinal detachment and proliferative vitreoretinopathy.

Background: In proliferative vitreoretinopathy (PVR), a nonangiogenic eye disease that is characterized by the formation of mainly avascular membranes, vascular endothelial growth factor (VEGF) levels are found to be upregulated. Recently, it was discovered that VEGF is alternatively spliced to form the angiogenic (VEGF xxx) and antiangiogenic (VEGF xxx b) family of isoforms. Previous studies on expression of VEGF in PVR samples have not distinguished between the two families of isoforms.

Levels of VEGF but not VEGF(165b) are increased in the vitreous of patients with retinal vein occlusion.

Purpose: To determine the concentration of the pro-angiogenic vascular endothelial growth factor VEGF(165) (VEGF) and the anti-angiogenic VEGF(165b) in vitreous samples of patients with branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO) in comparison to patients without retinal occlusive disease.

Design: Experimental laboratory investigation.

Methods: Vitreous samples were collected from patients undergoing surgery for arteriovenous dissection after BRVO, radial optic neurotomy after CRVO in the occlusion group, or macular pucker or macular hole in the control group.

Recombinant human VEGF165b inhibits experimental choroidal neovascularization.

Purpose: Vascular endothelial growth factor (VEGF-A) is the principal stimulator of angiogenesis in wet age-related macular degeneration (AMD). However, VEGF-A is generated by alternate splicing into two families, the proangiogenic VEGF-A(xxx) family and the antiangiogenic VEGF-A(xxx)b family. It is the proangiogenic family that is responsible for the blood vessel growth seen in AMD.

VEGF-A165b is cytoprotective and antiangiogenic in the retina.

Purpose: A number of key ocular diseases, including diabetic retinopathy and age-related macular degeneration, are characterized by localized areas of epithelial or endothelial damage, which can ultimately result in the growth of fragile new blood vessels, vitreous hemorrhage, and retinal detachment. VEGF-A(165), the principal neovascular agent in ocular angiogenic conditions, is formed by proximal splice site selection in its terminal exon 8. Alternative splicing of this exon results in an antiangiogenic isoform, VEGF-A(165)b, which is downregulated in diabetic retinopathy.

Regulation of vascular endothelial growth factor (VEGF) splicing from pro-angiogenic to anti-angiogenic isoforms: a novel therapeutic strategy for angiogenesis.

Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF(165), and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF(165)b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice.

VEGF(121)b, a new member of the VEGF(xxx)b family of VEGF-A splice isoforms, inhibits neovascularisation and tumour growth in vivo.

Background: The key mediator of new vessel formation in cancer and other diseases is VEGF-A. VEGF-A exists as alternatively spliced isoforms - the pro-angiogenic VEGF(xxx) family generated by exon 8 proximal splicing, and a sister family, termed VEGF(xxx)b, exemplified by VEGF(165)b, generated by distal splicing of exon 8. However, it is unknown whether this anti-angiogenic property of VEGF(165)b is a general property of the VEGF(xxx)b family of isoforms.

Therapeutic potential of manipulating VEGF splice isoforms in oncology.

Anti-angiogenic therapies currently revolve around targeting vascular endothelial growth factor-A (VEGF-A) or its receptors. These therapies are effective to some degree, but have low response rates and poor side-effect profiles. Part of these problems is likely to be due to their lack of specificity between pro- and anti-angiogenic isoforms, and their nonspecific effects on proactive, pleiotropic survival and maintenance roles of VEGF-A in endothelial and other cell types.

Transcriptional profiling reveals a critical role for tyrosine phosphatase VE-PTP in regulation of VEGFR2 activity and endothelial cell morphogenesis.

To define molecular events accompanying formation of the 3-dimensional (3D) vascular tube, we have characterized gene expression during vascular endothelial growth factor (VEGF)-induced tubular morphogenesis of endothelial cells. Microarray analyses were performed comparing gene induction in growth-arrested, tube-forming endothelial cells harvested from 3D collagen cultures to that in proliferating endothelial cells cultured on fibronectin. Differentially expressed genes were clustered and analyzed for specific endothelial expression through publicly available datasets.

Recombinant human VEGF165b protein is an effective anti-cancer agent in mice.

Tumour growth is dependent on angiogenesis, the key mediator of which is vascular endothelial growth factor-A (VEGF-A). VEGF-A exists as two families of alternatively spliced isoforms - pro-angiogenic VEGF(xxx) generated by proximal, and anti-angiogenic VEGF(xxx)b by distal splicing of exon 8. VEGF(165)b inhibits angiogenesis and is downregulated in tumours.

VEGF 165 b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy.

Bevacizumab, an anti-vascular endothelial growth factor (VEGF-A) antibody, is used in metastatic colorectal carcinoma (CRC) treatment, but responses are unpredictable. Vascular endothelial growth factor is alternatively spliced to form proangiogenic VEGF(165) and antiangiogenic VEGF(165)b. Using isoform-specific enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, we found that over 90% of the VEGF in normal colonic tissue was VEGF(xxx)b, but there was a variable upregulation of VEGF(xxx) and downregulation of VEGF(xxx)b in paired human CRC samples.

The endogenous anti-angiogenic VEGF isoform, VEGF165b inhibits human tumour growth in mice.

Vascular endothelial growth factor-A is widely regarded as the principal stimulator of angiogenesis required for tumour growth. VEGF is generated as multiple isoforms of two families, the pro-angiogenic family generated by proximal splice site selection in the terminal exon, termed VEGFxxx, and the anti-angiogenic family formed by distal splice site selection in the terminal exon, termed VEGFxxxb, where xxx is the amino acid number. The most studied isoforms, VEGF165 and VEGF165b have been shown to be present in tumour and normal tissues respectively.

Endocan is a VEGF-A and PI3K regulated gene with increased expression in human renal cancer.

An in vitro model of VEGF-A-induced angiogenesis was used to generate transcription profiles of human microvascular endothelial cells. Microarray analysis showed increased transcription of genes known to regulate angiogenesis, but also genes that previously have not been firmly associated with angiogenesis such as endocan, pinin, plakophilin, phosphodiesterase 4B and gelsolin. Increased endocan mRNA levels in response to VEGF-A in endothelial cells and in human renal cancer have previously been reported.

Regulation of endothelial cell differentiation and transformation by H-Ras.

Angiogenesis is regulated by growth factors which activate tyrosine kinase receptors leading to the activation of a number of intracellular signaling pathways. The specific function of H-Ras during FGF-2 stimulated endothelial cell differentiation, defined as invasive growth and formation of branching networks in fibrin gels, was investigated by using conditionally immortalized endothelial cell lines induced to express H-Ras mutants. Expression of inhibitory N17Ras did not impair differentiation in response to FGF-2 and TNF-alpha.

How to make tetracycline-regulated transgene expression go on and off.

Tetracycline-regulated gene expression systems are widely used to allow temporal and quantitative control of transgene expression in cultured cells and transgenic animals. While working with the Tet-Off system, where tetracycline or the analogue doxycycline suppresses expression, we noted a considerable variability in induced transgene expression after removal of doxycycline. Variable expression of the transgene could not be explained by clonal variation since it was noted when working with clonal cell lines.

Homogeneous scoring of single-nucleotide polymorphisms: comparison of the 5'-nuclease TaqMan assay and Molecular Beacon probes.

Homogeneous assays based on real-time fluorescence monitoring during PCR are relevant alternatives for large-scale genotyping of single-nucleotide polymorphisms (SNPs). We compared the performance of the homogeneous TaqMan 5'-nuclease assay and the Molecular Beacon assay using three SNPs in the human estrogen receptor gene as targets. When analyzing a panel of 90 DNA samples, both assays yielded a comparable power of discrimination between the genotypes of a C-to-T transition in codon 10 and a G-to-A transition in codon 594 of the estrogen receptor gene.