Differential regulation of blood flow-induced neovascularization and mural cell recruitment by vascular endothelial growth factor and angiopoietin signalling.

KEY POINTS: Combining nitric oxide (NO)-mediated increased blood flow with angiopoietin-1-Tie2 receptor signalling induces arteriolargenesis - the formation of arterioles from capillaries - in a model of physiological angiogenesis. This NO-Tie-mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling. Inhibition of VEGF signalling increases pericyte coverage in microvessels. Together these findings indicate that generation of functional neovasculature requires close titration of NO-Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful. ABSTRACT: Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains-2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin-1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)-mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over-expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms-like tyrosine kinase receptor-1 (sFlt1). We found that NO-mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin-1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte recruitment to newly formed vessels (from 27 ± 2 to 54 ± 3% pericyte ensheathment). We demonstrate that a fine balance of VEGF and angiopoietin signalling is required for the formation of a functional vascular network. Endogenous VEGF signalling prevents excess neovessel pericyte coverage, and is required for VSMC recruitment during increased nitric oxide-mediated vasodilatation and angiopoietin signalling (NO-Tie-mediated arteriogenesis). Therapeutic vascular remodelling paradigms may therefore require treatments that modulate blood flow to utilize endogenous VEGF, in combination with exogenous Ang1, for effective neovascularization.

United Kingdom health research analyses and the benefits of shared data.

BACKGROUND: To allow research organisations to co-ordinate activity to the benefit of national and international funding strategies requires assessment of the funding landscape; this, in turn, relies on a consistent approach for comparing expenditure on research. Here, we discuss the impact and benefits of the United Kingdom's Health Research Classification System (HRCS) in national landscaping analysis of health research and the pros and cons of performing large-scale funding analyses. METHODS: The first United Kingdom health research analysis (2004/2005) brought together the 11 largest public and charity funders of health research to develop the HRCS and use this categorisation to examine United Kingdom health research. The analysis was revisited in 2009/2010 and again in 2014. The most recent quinquennial analysis in 2014 compiled data from 64 United Kingdom research organisations, accounting for 91% of all public/charitable health research funding in the United Kingdom. The three analyses summarise the United Kingdom's health research expenditure in 2004/2005, 2009/2010 and 2014, and can be used to identify changes in research activity and disease focus over this 10 year period. RESULTS: The 2004/2005 analysis provided a baseline for future reporting and evidence for a United Kingdom Government review that recommended the co-ordination of United Kingdom health research should be strengthened to accelerate the translation of basic research into clinical and economic benefits. Through the second and third analyses, we observed strategic prioritisation of certain health research activities and disease areas, with a strong trend toward increased funding for more translational research, and increases in specific areas such as research on prevention. CONCLUSIONS: The use of HRCS in the United Kingdom to analyse the research landscape has provided benefit both to individual participatory funders and in coordinating initiatives at a national level. A modest amount of data for each project is sufficient for a nationwide assessment of health research funding, but achieving coverage of the United Kingdom portfolio relies on sourcing these details from a large number of individual funding agencies. The effort needed to compile this data could be minimised if funders routinely shared or published this information in a standard and accessible way. The United Kingdom approach to landscaping analyses could be readily adapted to suit other groups or nations, and global availability of research funding data would support better national and international coordination of health research.

Circulating levels of anti-angiogenic VEGF-A isoform (VEGF-Axxxb) in colorectal cancer patients predicts tumour VEGF-A ratios.

PURPOSE: Bevacizumab as an adjunct to chemotherapy improves survival for some patients with metastatic colorectal cancer. Immunohistochemical staining of samples from the registration ECOG E3200 trial of bevacizumab with FOLFOX demonstrated that only patients with carcinomas expressing low levels of VEGF-A165b, an anti-angiogenic splice variant of the Vascular Endothelial Growth Factor family of proteins, benefited from bevacizumab treatment. To identify a more useful biomarker of response we tested the hypothesis that circulating VEGF-A165b levels correlate with immunohistochemical staining. EXPERIMENTAL DESIGN: 17 patients with biopsy proven colorectal adenocarcinoma had pre-operative blood samples drawn. They underwent resection and had post-resection blood drawn. The plasma was analysed for levels of VEGF-Axxxb using enzyme-linked immunosorbent assay (ELISA) and the tumour blocks stained for VEGF-Axxxb and pan-VEGF-A. The normalised ratio of VEGF-Axxxb expression to that of panVEGF-A expression scored by IHC was calculated and correlated with plasma VEGF-A165b levels. RESULTS: Plasma levels of VEGF-Axxxb significantly correlated with the VEGF-Axxxb:panVEGF-A ratio (r=0.594, P<0.02) in colorectal cancers. Median plasma VEGF-Axxxb levels were 151 pg/ml. The mean (1.5±0.17) and median, IQR (1.8, 1-2) IHC scores of the patients with greater than median plasma VEGF-Axxxb were significantly greater than those with less than median plasma VEGF-Axxxb levels (mean ± SEM=0.85±10.12, median, IQR=1, 0.54-1). CONCLUSION: These results suggest that plasma VEGF-Axxxb levels could be an effective biomarker of response to Bevacizumab. These results indicate that a prospective trial is warranted to explore the use of plasma VEGF-Axxxb levels to stratify patients for colorectal cancer treatment by bevacizumab.

The carboxyl terminus of VEGF-A is a potential target for anti-angiogenic therapy.

Anti-VEGF-A therapy has become a mainstay of treatment for ocular neovascularisation and in cancer; however, their effectiveness is not universal, in some cases only benefiting a minority of patients. Anti-VEGF-A therapies bind and block both pro-angiogenic VEGF-Axxx and the partial agonist VEGF-Axxxb isoforms, but their anti-angiogenic benefit only comes about from targeting the pro-angiogenic isoforms. Therefore, antibodies that exclusively target the pro-angiogenic isoforms may be more effective. To determine whether C-terminal-targeted antibodies could inhibit angiogenesis, we generated a polyclonal antibody to the last nine amino acids of VEGF-A165 and tested it in vitro and in vivo. The exon8a polyclonal antibody (Exon8apab) did not bind VEGF-A165b even at greater than 100-fold excess concentration, and dose dependently inhibited VEGF-A165 induced endothelial migration in vitro at concentrations similar to the VEGF-A antibody fragment ranibizumab. Exon8apab can inhibit tumour growth of LS174t cells implanted in vivo and blood vessel growth in the eye in models of age-related macular degeneration, with equal efficacy to non-selective anti-VEGF-A antibodies. It also showed that it was the VEGF-Axxx levels specifically that were upregulated in plasma from patients with proliferative diabetic retinopathy. These results suggest that VEGF-A165-specific antibodies can be therapeutically useful.

Detection of VEGF-A(xxx)b isoforms in human tissues.

Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.

ß-Defensin 1 haplotype associated with postoperative endophthalmitis.

PURPOSE: Endophthalmitis is a rare but sight-threatening complication of intraocular surgery. ß-Defensins are antimicrobial peptides that appear to be important components of the ocular immune response. We propose that variation in defensin genes may alter susceptibility to endophthalmitis. METHODS: Post-cataract endophthalmitis patients (n = 28) and post-cataract controls (n = 75) were recruited and DNA samples extracted. The ß-defensin 1 gene (DEFB1) was screened for single-nucleotide polymorphisms (SNPs) using bidirectional sequencing. Case-control statistical assessment was undertaken for both the individual polymorphic loci observed and combined haplotypes using PHASE software. RESULTS: We identified 19 SNPs and observed strong linkage disequilibrium within the gene. We found that the three-SNP haplotype -688C/-44C/-20A was associated strongly with endophthalmitis [odds ratio (OR) = 8.88 (1.74, 45.42), corrected p = 0.0095]. Furthermore, we uncovered several trends, including increased prevalence of the -44CC genotype in the endophthalmitis group. CONCLUSION: We have shown previously that the -44CC SNP genotype was present in a single case of bilateral endophthalmitis. In this study, we found this genotype to be more common in the endophthalmitis group and a mini-haplotype including this SNP was associated strongly with endophthalmitis. There is functional evidence that this genetic profile decreases transcription of the ß-defensin 1 peptide and could therefore reduce the innate ocular immune defence.

VEGF polymorphisms are associated with severity of diabetic retinopathy.

PURPOSE: To determine whether single nucleotide polymorphisms (SNPs) in the vascular endothelial growth factor (VEGF) gene are associated with severity of diabetic retinopathy. METHODS: A case-control study was conducted in which 45 individuals with type 1 or 2 diabetes with proliferative diabetic retinopathy (PDR) and 61 individuals with type 1 or 2 diabetes without retinopathy (DWR) were genotyped for 14 SNPs in the VEGF promoter and gene. RESULTS: Three of the promoter SNP genotypes, -160C, -152A (rs13207351), and -116A (rs1570360), showed significant independent associations with PDR, as well as the minihaplotype CAA (P = 0.00017). Two promoter haplotypes were associated with severity of retinopathy: -460C, -417T, -172C, -165C, -160C, -152A, -141A, -116A, +405C was associated with PDR (OR [95% CI] = 29.92 [3.91, 228.78], P = 1.62 x 10(-5)) and -460C, -2417T, -172C, -165C, -160C, -152A, -141A, -116G, +405G was associated with DWR (OR = 0.05 [0.01, 0.35], P = 0.000373). Furthermore, two haplotype-tagged (ht) SNPs, +4618 (rs735286) and +5092 (rs2146323), and five htSNP haplotypes were associated with severity of retinopathy. When the nine promoter/5' untranslated region [UTR] and five htSNP genotypes were combined into a 14-SNP haplotype, a single haplotype, -460C, -417T, -172C, -165C, -160C, -152A, -141A, -116A, +405C, +674T, +4618C, +5092A, +9162C, +9512C was found to be significantly associated with the PDR group (OR = 18.45 [2.35, 144.67], P = 0.00622). CONCLUSIONS: A clear association was demonstrated between VEGF SNPs and severity of diabetic retinopathy. Furthermore, two of the htSNP haplotypes appear to be more generalized markers for angiogenesis, in that these have been found in prior work to be associated with neovascular age-related macular degeneration.

VEGF polymorphisms are associated with neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common cause of blindness in the elderly. Linkage has been shown to the vascular endothelial growth factor (VEGF) gene and ocular levels of VEGF are raised in individuals with the neovascular form of disease. To examine the role of VEGF further, we conducted a case-control study where 45 individuals with neovascular AMD and 94 age-matched controls were genotyped for 14 single nucleotide polymorphisms (SNPs) in the VEGF promoter and gene. The single SNP +674 CC genotype was significantly associated with AMD (OR=2.40, 95%CI 1.09-5.26, P=0.027). Haplotype analysis of SNPs +674, +4618, +5092, +9162 and +9512 revealed that CTCCT and TCACC were associated with AMD (OR=15.77, 95% CI 1.91-130.24, P=0.0161 and OR=9.95, 95%CI 3.22-30.74, P=0.000053, respectively). The haplotype TCACT was associated with the control group (P=0.0001832). Furthermore, haplotype analysis of promoter SNPs revealed that possession of the -460T, -417T, -172C, -165C, -160C, -152G, -141A, -116A, +405C haplotype was strongly associated with AMD (OR=18.24, 95%CI 2.25-148.25, P=0.0074). This is the most extensive analysis of the VEGF gene in AMD, demonstrating a clear association with the exudative form of disease, thereby creating the possibility for predictive testing. Smoking, high fat intake and hypertension are negative environmental risk factors in AMD, whereas increased consumption of dietary antioxidants can have a protective effect. Identification of those at risk in the population would allow individual counselling with lifestyle advice to reduce the risks of blindness. (Genbank accession nos M63971 and AF437895).