James G. Carter

Detection of VEGF-Axxxb 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.

Analysis of SERPING1 and its association with age-related macular degeneration

Purpose The genetics of age-related macular degeneration (AMD) pathogenesis is a growing area of interest. In the last year a new potential association locus has been reported; SERPING1, a C1 inhibitor of the compliment pathway. We present our own assessment of the SERPING1 AMD-risk locus. Methods Patients with exudative AMD (n=94) and age-matched controls (n=95) were selected for study. All subjects were genotyped for the SERPING1 polymorphism identified as a new risk locus for AMD; rs2511989, IVS6-865 g>a. Genotyping was determined by direct sequencing. Statistical analysis was attained using SPSS v15 (SPSS Inc.). Results Carriage of the G allele was raised in AMD group vs. controls (61.2% vs. 57.6%), but was not statistically significant (p=0.48). No significant associations were seen in genotype carriage between AMD and control groups (e.g. GG genotype, 40.4% vs. 30.4%, p=0.22). Conclusion No significant associations were seen between SERPING1 and AMD in this study. Only two published analyses have examined this potential association, and these studies produced conflicting results. While it appears SERPING1 mRNA is produced in the eye, the functional effects of the rs2511989 locus have yet to be determined. More work is needed to identify the biological role, if any, SERPING1 plays in the development of AMD.

Splicing factor polymorphisms, the control of VEGF isoforms and association with angiogenic eye disease.

PURPOSE Alternative splicing of the last exon (exon 8) of vascular endothelial growth factor (VEGF) pre-mRNA is a key element in the balance of pro- and anti-angiogenic VEGF isoforms in exudative age-related macular degeneration (exAMD) and proliferative diabetic retinopathy (PDR). Three splicing factors, SRp40, ASF/SF2, and SRp55 are predicted to control alternative splicing by binding to exonic splice enhancers (ESE) in VEGF exon 8. This pilot study examines whether there is an association between angiogenic eye disease and splicing factor polymorphisms, and whether there are sequence variations in the alternative splice sites of the VEGF gene. MATERIALS AND METHODS A case:control pilot study comparing 163 individuals with angiogenic eye disease (94 exAMD and 69 PDR patients) with 95 age-matched controls. Splicing factor polymorphisms were genotyped by Restriction Fragment Length Polymorphism (RFLP) and sequencing, and the VEGF alternatively spliced region was assessed by denaturing High Performance Liquid Chromatography (dHPLC) using a transgenomic WAVE heteroduplex analyzer. RESULTS No variations were observed in the alternatively spliced region of VEGF exon 8. ASF/SF2 polymorphisms showed no association with exAMD or PDR. For PDR, we observed a trend in SRp40 (rs6573908) where the 5136CC genotype was more frequent in controls (p = 0.0517) and a significant association of the SRp55 (rs2235611), where the 2994C allele was more common in the PDR group (p = 0.03). This remained strong, but not significant, after logistic regression for age, sex, disease type, and duration (p = 0.06). CONCLUSIONS The lack of variation in the VEGF alternatively spliced region suggests the importance of sequence conservation in this area in maintaining the balance of pro- and anti-angiogenic VEGF isoforms. The link between PDR and the SRp55 2994 polymorphism suggests a disease-specific association between factors controlling VEGF splicing and ocular angiogenesis.

β‐Defensin 1 haplotype associated with postoperative endophthalmitis

Acta Ophthalmol. 2010: 88: 786–790

Differential regulation of blood flow induced neovascularisation and mural cell recruitment by VEGF and angiopoietin signalling

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.

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

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.

A case of bilateral endophthalmitis and carriage of β-defensin 1 –44CC genotype

β-defensins are innate antimicrobial peptides found in a variety of ocular tissues and are critical to the immune response.1–3 We present a case of bilateral endophthalmitis and show the carriage of the –44CC genotype in β-defensin 1. The polymorphism has previously been linked to increased susceptibility to infection.4–7 View this table: Table 1 Sequence variations seen in β-defensin 1 and β-defensin 2 genes in a patient with bilateral endophthalmitis An 80-year-old man underwent cataract surgery in the right eye complicated by a capsular tear. Within 4 days he returned with a hypopyon, painful loss of vision and reddening of the right eye. Endophthalmitis was diagnosed, an urgent vitreous tap was performed and intravitreal cetazidime and vancomycin were started. Microscopical examination revealed Gram-positive cocci, but no final growth of organisms. The right eye responded to treatment, but 1 month later a retinal detachment was diagnosed in the same eye. This was surgically repaired without complications. A year later, after an uncomplicated left cataract surgery in another hospital, the patient presented again with endophthalmitis symptoms, including hypopyon, rapid visual loss (hand movements), pain and reddening of the …