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.g., PSA, CA125, CA19-9, CEA, AFP) are frequently found in plasma at very low concentration (pg/mL-ng/mL). New sensitive and specific assays are therefore urgently required to detect the disease at an early stage when prognosis is good following surgical resection. This study was designed to meet the longstanding unmet clinical need for earlier CRC detection by measuring plasma candidate biomarkers of cancer onset and progression in a clinical stage-specific manner. EDTA plasma samples (1 µL) obtained from 75 patients with Dukes' staged CRC or unaffected controls (age and sex matched with stringent inclusion/exclusion criteria) were assayed for expression of 92 human proteins employing the Proseek® Multiplex Oncology I proximity extension assay. An identical set of plasma samples were analyzed utilizing the Bio-Plex Pro™ human cytokine 27-plex immunoassay. RESULTS: Similar quantitative expression patterns for 13 plasma antigens common to both platforms endorsed the potential efficacy of Proseek as an immune-based multiplex assay for proteomic biomarker research. Proseek found that expression of Carcinoembryonic Antigen (CEA), IL-8 and prolactin are significantly correlated with CRC stage. CONCLUSIONS: CEA, IL-8 and prolactin expression were found to identify between control (unaffected), non-malignant (Dukes' A + B) and malignant (Dukes' C + D) stages.

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. METHODS: We measured total VEGF and VEGF xxx b levels in subretinal fluid of patients with PVR (n = 10) and in patients with uncomplicated rhegmatogenous retinal detachment (n = 27) using enzyme-linked immunosorbent assay. RESULTS: : We found total VEGF levels to be 2- to 3-fold elevated in the PVR group as compared with the rhegmatogenous retinal detachment group (P = 0.047). Antiangiogenic VEGF xxx b isoforms predominated (>60% of total VEGF) in the majority of rhegmatogenous retinal detachment and PVR samples investigated, although a wide variability of isoform ratios was observed within both groups. CONCLUSION: The absence of an increased ratio of VEGF xxx to VEGF xxx b in patients with PVR as compared with patients with uncomplicated rhegmatogenous retinal detachment may explain a lack of blood vessels in PVR membranes. Elevated VEGF levels indicate that this cytokine may play a role in the pathogenesis of PVR that is not related to angiogenesis.

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. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF(165) and less VEGF(165)b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.

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. RESULTS: Human anti-Ang2 and anti-angiopoietin1(Ang1)/Ang2 antibodies blocked colorectal carcinoma growth in immuno-compromised mice (p < 0.001, n = 6). Injection of 1 µg of Ang2 or Ang2/Ang1 antibody-inhibited angiogenesis in models of retinal (p < 0.001, n = 6), and choroidal neovascularization (p < 0.001, n = 11-13 per group) to levels similar to that with anti-VEGF antibodies. There was no difference between Ang2 specific and Ang1/Ang2 bi-specific antibodies. In vitro, Ang2 antibodies showed no cytotoxicity and did not inhibit endothelial cell migration or proliferation. CONCLUSION: Thus, human Ang2 antibodies are potentially therapeutic agents for ocular neovascularization in models of retinal and choroidal neovascularization, in the absence of VEGF inhibition.

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. We validated the contribution of one of the identified genes, vascular endothelial protein tyrosine phosphatase (VE-PTP), to endothelial morphogenesis. Silencing of VE-PTP expression was accompanied by increased VEGF receptor-2 (VEGFR2) tyrosine phosphorylation and activation of downstream signaling pathways. The increased VEGFR2 activity promoted endothelial cell cycle progression, overcoming the G(0)/G(1) arrest associated with organization into tubular structures in the 3D cultures. Proximity ligation showed close association between VEGFR2 and VE-PTP in resting cells. Activation of VEGFR2 by VEGF led to rapid loss of association, which was resumed with time in parallel with decreased receptor activity. In conclusion, we have identified genes, which may serve critical functions in formation of the vascular tube. One of these, VE-PTP, regulates VEGFR2 activity thereby modulating the VEGF-response during angiogenesis.

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. An alternative approach, and one which has recently been shown to be effective in animal models of neovascularization in the eye, is to target the mechanisms by which the cell generates pro-angiogenic splice forms of VEGF-A, its receptors and, co-incidentally, by targeting the upstream processes, other oncogenes that have antagonistic splice isoforms. The concept here is to target the splicing mechanisms that control splice site choice in the VEGF-A mRNA. Recent evidence on the pharmacological possibilities of such splice factors is described.

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. Concentrations of VEGF and VEGF(165b) were determined by ELISA and an ELISA-type antibody microarray. RESULTS: Average vitreal concentration of VEGF was 8.6 ng/mL in the CRVO group and 2.0 ng/mL in the BRVO group as compared to 0.26 ng/mL in the control group. Average vitreal concentration of VEGF(165b) was 27 pg/mL in the CRVO group, 42 pg/mL in the BRVO group, and 49 pg/mL in the control group. In patients with CRVO and BRVO, the angiogenic balance was shifted towards angiogenic stimulation. CONCLUSION: The severity of RVO from BRVO to CRVO correlates with an increase of VEGF and the decrease of VEGF(165b), indicating a pro-angiogenic shift. Altering the ratio of VEGF(165b)/VEGF(165) might be a feasible approach for treating retinal occlusive diseases.

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. WT1 bound to the SRPK1 promoter, and repressed expression through a specific WT1 binding site. In WT1 mutant cells SRPK1-mediated hyperphosphorylation of the oncogenic RNA binding protein SRSF1 regulated splicing of VEGF and rendered WT1 mutant cells proangiogenic. Altered VEGF splicing was reversed by wild-type WT1, knockdown of SRSF1, or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumor growth.

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. METHODS: To determine the role of antiangiogenic isoforms of VEGF-A as inhibitors of choroidal neovascularization, the authors used a model of laser-induced choroidal neovascularization in the mouse eye and investigated VEGF-A(165)b effects on endothelial cells and VEGFRs in vitro. RESULTS: VEGF-A(165)b inhibited VEGF-A(165)-mediated endothelial cell migration with a dose effect similar to that of ranibizumab and bevacizumab and 200-fold more potent than that of pegaptanib. VEGF-A(165)b bound both VEGFR1 and VEGFR2 with affinity similar to that of VEGF-A(165). After laser injury, mice were injected either intraocularly or subcutaneously with recombinant human VEGF-A(165)b. Intraocular injection of rhVEGF-A(165)b gave a pronounced dose-dependent inhibition of fluorescein leakage, with an IC(50) of 16 pg/eye, neovascularization (IC(50), 0.8 pg/eye), and lesion as assessed by histologic staining (IC(50), 8 pg/eye). Subcutaneous administration of 100 microg twice a week also inhibited fluorescein leakage and neovascularization and reduced lesion size. CONCLUSIONS: These results show that VEGF-A(165)b is a potent antiangiogenic agent in a mouse model of age-related macular degeneration and suggest that increasing the ratio of antiangiogenic-to-proangiogenic isoforms may be therapeutically effective in this condition.

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. Here the authors investigate the antiangiogenic activity of VEGF(165)b and its effect on retinal epithelial and endothelial cell survival. METHODS: VEGF-A(165)b was injected intraocularly in a mouse model of retinal neovascularization (oxygen-induced retinopathy [OIR]). Cytotoxicity and cell migration assays were used to determine the effect of VEGF-A(165)b. RESULTS: VEGF-A(165)b dose dependently inhibited angiogenesis (IC(50), 12.6 pg/eye) and retinal endothelial migration induced by 1 nM VEGF-A(165) across monolayers in culture (IC(50), 1 nM). However, it also acts as a survival factor for endothelial cells and retinal epithelial cells through VEGFR2 and can stimulate downstream signaling. Furthermore, VEGF-A(165)b injection, while inhibiting neovascular proliferation in the eye, reduced the ischemic insult in OIR (IC(50), 2.6 pg/eye). Unlike bevacizumab, pegaptanib did not interact directly with VEGF-A(165)b. CONCLUSIONS: The survival effects of VEGF-A(165)b signaling can protect the retina from ischemic damage. These results suggest that VEGF-A(165)b may be a useful therapeutic agent in ischemia-induced angiogenesis and a cytoprotective agent for retinal pigment epithelial cells.

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. Here, we show for the first time that administration of recombinant human VEGF(165)b inhibits colon carcinoma tumour growth and tumour vessel density in nude mice, with a terminal plasma half-life of 6.2h and directly inhibited angiogenic parameters (endothelial sprouting, orientation and structure formation) in vitro. Intravenous injection of (125)I-VEGF(165)b demonstrated significant tumour uptake lasting at least 24h. No adverse effects on liver function or haemodynamics were observed. These results indicate that injected VEGF(165)b was taken up into the tumour as an effective anti-angiogenic cancer therapy, and provide proof of principle for the development of this anti-angiogenic growth factor splice isoform as a novel cancer therapy.

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. We now show increased endocan protein levels in VEGF-A treated endothelial cells and in human renal clear cell carcinoma. Increased protein expression was observed both in tumor cells and in a subset of tumor vessels, while expression in normal kidney tissue was low. VEGF-A seemed to be a specific inducer of endocan transcription since FGF-2, PDGF-BB, HGF/SF and EGF did not alter expression levels. Inhibition of PI3K with LY294002 caused a 12-fold increase in endocan transcription suggesting a repressive function of PI3K. In contrast inhibition of Src or MEK, which are signaling pathways activated by VEGF-A, did not influence basal or VEGF-A-induced endocan levels. In conclusion our study shows that, among angiogenic growth factors, VEGF-A is a specific inducer of endocan transcription which is translated into increased protein levels in VEGF-A treated endothelial cells. Increased endocan protein expression in human renal cancer suggests a role in tumor growth.

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. Instead we found that doxycycline bound nonspecifically to cells and extracellular matrix and was slowly released after it had been removed from tissue culture media. The released doxycycline reached sufficiently high levels to completely suppress transgene expression. The effect was not dependent on cell type or the nature of the transgene. However, robust and rapid transgene expression could be induced if released doxycycline were removed by washing cells 3h after the initial removal of doxycycline. The use of different vector systems, harboring the tetracycline-regulatable components, yielded similar results. These results not only help explain why tetracycline-regulatable transgene expression systems sometimes are variable but also provide simple ways to substantially improve the efficiency, utility, and reliability of these widely used expression systems.

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. The farnesyltransferase inhibitor FTI-277 inhibited farnesylation of Ras but did not inhibit differentiation of human microvascular endothelial cells or mouse brain endothelial cells. In contrast, activated V12Ras inhibited endothelial cell differentiation and cells displayed a transformed phenotype with an increased rate of proliferation and loss of contact inhibited growth. Furthermore, V12Ras expressing endothelial cells grew as solid tumors when injected subcutaneously into mice. Our data suggest that, in endothelial cells, H-Ras activity is not required for differentiation. However, this activity must be tightly regulated as aberrant activity can disturb the ability of endothelial cells to undergo differentiation.