Diabetic retinopathy is associated with a switch in splicing from anti- to pro-angiogenic isoforms of vascular endothelial growth factor
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- Perrin, R.M., Konopatskaya, O., Qiu, Y. et al. Diabetologia (2005) 48: 2422. doi:10.1007/s00125-005-1951-8
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Proliferative diabetic retinopathy results from excess blood vessel growth into the vitreous fluid of the eye. Retinal angiogenesis is regulated by expression of vascular endothelial growth factor (VEGF), and many studies have shown that VEGF is critically involved in proliferative diabetic retinopathy. VEGF is alternatively spliced to form the angiogenic (VEGFxxx) and potentially anti-angiogenic (VEGFxxxb) family of isoforms. The VEGFxxxb family is found in normal tissues, but down-regulated in renal and prostate cancer. Previous studies on endogenous expression of VEGF in the eye have not distinguished between the two families of isoforms.
We measured VEGFxxxb isoform expression in normal human eye tissue (lens, sclera, retina and iris) and vitreous fluid using enzyme-linked immunosorbent assay and Western blotting with a VEGFxxxb-specific antibody.
VEGFxxxb protein was expressed in lens, sclera, retina, iris and vitreous fluid. Multiple isoforms were seen, including VEGF165b, VEGF121b, VEGF145b, VEGF183b and VEGF189b. In non-diabetic patients, 64±7% of the VEGF in the vitreous was VEGFxxxb (n=18), whereas in diabetic patients only 12.5±3.6% of total VEGF was VEGFxxxb.
Since VEGFxxxb inhibits VEGFxxx-induced angiogenesis in a one-to-one stoichiometric manner, these results show that in the eye of diabetic patients VEGF splicing was switched from an anti-angiogenic to a pro-angiogenic environment. This occurred through changes to the ratio of VEGFxxx : VEGFxxxb. Alterations to splicing, and through that to the balance of VEGF isoforms, could therefore be a potential therapeutic strategy for diabetic retinopathy.
KeywordsAngiogenesis Proliferative diabetic retinopathy Splicing VEGF VEGF121b VEGF165b
glyceraldehyde-3-phosphate dehydrogenase gene
vascular endothelial growth factor
angiogenic isoform of VEGF
anti-angiogenic isoform of VEGF
Diabetic retinopathy is the leading cause of blindness in the working population of the Western world and one of the most prominent pathological complications of diabetes. In the early stages the dominant features are microaneurysms, small haemorrhages and leakage of lipids into the retina. With time this progresses to capillary closure, retinal hypoxia and more extensive exudation from retinal vessels. Advanced retinopathy is reached when excessive capillary closure has led to abnormal proliferation of retinal vessels, accompanied by vitreous haemorrhage and fibrosis . The development of retinal neovascularisation is obligatorily dependent on hypoxia-induced production of vascular growth factors, and this is thought to be a major contributor to diabetic retinal neovascularisation and proliferative retinopathy . This hypoxia-driven angiogenesis is a complex process mediated by factors from the vascular endothelial growth factor (VEGF), angiopoietin and ephrin families. Angiogenesis underlies many other disease states, including cancer, rheumatoid arthritis and psoriasis . New therapies that influence new vessel formation are effective in animals and have entered clinical trials in retinopathy as well as in cancer and heart disease. VEGF (also referred to as VEGF-A) is the dominant pro-angiogenic factor in diabetic retinopathy, stimulating endothelial cell proliferation, migration and increased microvascular permeability and blood flow by activation of its cognate receptors VEGF-receptor 1 (FLT1) and VEGF-R2 (KDR/FLK1) .
VEGF has been shown to be significantly up-regulated in diabetic retinopathy, particularly in retinal pigmented epithelial cells, glial cells and vitreal fibroblasts . Furthermore, there is mounting functional evidence that inhibition of angiogenesis, both non-specifically with pharmacological anti-angiogenic agents and specifically with anti-VEGF agents, ameliorates neovascularisation in well-characterised animal models of retinopathy . Subtle mutations in the 5′ region of the VEGF gene can predispose to diabetic retinopathy, suggesting that regulation of transcription or splicing of the VEGF gene is important in this condition . Recent clinical trials of agents that inhibit VEGF have been shown to have some effectiveness in choroidal neovascularisation cause by age-related macular degeneration  and in animal models of retinal neovascularisation .
The first member of this family to be identified was VEGF165b, and this is the only isoform which has been characterised in terms of its actions on endothelial cells. The receptor-binding domains are still present in VEGF165b; it binds to the receptor but does not stimulate angiogenesis signalling. Furthermore, it inhibits the proliferative, migratory and vasodilator effects of VEGF165 and, unlike the VEGFxxx family of isoforms, VEGFxxxb is down-regulated in both renal cell carcinoma and malignant prostate tissue [8, 9]. Neither VEGFxxxb expression in the eye, nor relative expression of the pro- versus anti-angiogenic isoforms in the diabetic eye have been investigated previously.
Materials and methods
All materials were provided by Sigma, unless otherwise specified. The VEGF165b antibody was generated in house as previously described  (clone 56/1), and is available from the corresponding author. The VEGF antibodies for Western blotting were from Santa Cruz Biotechnology (VEGF-A20 antibody; Santa Cruz, CA, USA). Capture antibody for ELISA, VEGF165b protein, VEGF165 protein and the Duoset VEGF ELISA detection kit were provided by R&D Systems (Minneapolis, MN, USA).
Vitreous fluid was collected from 13 diabetic patients undergoing vitrectomy for complicated proliferative retinopathy. Three of these had type 1, the remainder type 2 diabetes. Control vitreous samples were taken from 18 patients without diabetes and with normal blood glucose, who were undergoing vitrectomy for various conditions, including retinal detachment and macular hole surgery. Human eye tissue was obtained from corneal graft donors within 48 h of death. All human tissue was collected with informed consent and local ethics committee approval.
Measurement of total VEGF and VEGFxxxb
Total VEGF concentrations in vitreous fluid were measured using a standard VEGF ELISA (Duoset). VEGFxxxb was measured with a similar capture ELISA, using the same capture antibody as in the pan-VEGF ELISA, but with a mouse monoclonal detection antibody raised in house against the terminal nine amino acids of VEGF165b, and a standard curve using recombinant human VEGF165b as described by Woolard and colleagues . A biotinylated secondary goat anti-mouse IgG was used to detect the VEGF165b antibody. To determine the concentration of protein in the vitreous, the concentration in the vitrectomy specimens was calculated assuming a total eye volume of 4 ml. Concentrations of VEGFxxx were calculated by subtracting VEGFxxxb from total VEGF.
Western blotting of membranes containing 12–15 μg of protein extracted from microdissected human eye tissues was carried out as previously described . Blots were probed only once, with either a pan-VEGF antibody or the VEGF165b antibody. Figures are representative blots from tissue taken from six donors, and probed separately.
Vitreous fluid was collected from consenting individuals undergoing surgery at Bristol Eye Hospital. These samples were kept on ice and RNA was extracted as soon as possible. The vitreous fluid was transferred to 50 ml Falcon tubes and spun at 2500 g (RC-3B; Sorvall, Stevenage, UK) for 10 min at 4°C, and the pellet was retained for RNA extraction. Each pellet was homogenised in 750 μl Trizol reagent and mRNA extracted according to the manufacturers’ instructions. Five per cent mRNA was reverse transcribed using poly d(T) as a primer and Expand RT (Roche, Indianapolis, IN, USA). This was then amplified by PCR using intron-spanning primers specific for VEGF165b, which specifically detect VEGF165b, even in the presence of a 50-fold greater concentration of VEGF165 . The primers were: antisense primer 5′-TCA GTC TTT CCT GGT GAG AGA TCT GCA-3′, sense primer 5′- TTG TAC AAG ATC CGC AGA CG -3′. The gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as a housekeeping gene, was amplified from each sample using sense primer 5′-GTC TTC ACC ACC ATG GAG-3′ and antisense primer 5′-ACC TTG CCC ACA GCC TTG GC-3′. cDNA was amplified using 1 μmol/l of each primer (20 pmol), 1.5 mmol/l MgCl2, 200 mmol/l dNTPs, and 2 units of Taq polymerase (AbGene, Epsom, Surrey, UK). Reactions were cycled 35 times (denaturing at 94°C for 30 s, annealing at 63°C for 30 s and extending at 72°C for 60 s). Products were analysed on a 3% agarose gel with 0.5 g/ml ethidium bromide. For semiquantitation, the gels were scanned into Adobe Photoshop using an Agfa scanner; the intensity of the band was measured with Image J (NIH, Bethesda, MD, USA) and normalised to the intensity of the GAPDH band.
VEGFxxxb isoforms are expressed in normal human eye tissue
VEGFxxxb mRNA isoforms are more detectable in normal than in diabetic vitreal cells
VEGFxxxb isoforms are the predominant isoform in normal but not in diabetic vitreous
Since the first identification of increased VEGF expression in the eyes of patients with diabetes by Adamis and colleagues in 1994, over 500 studies have investigated VEGF in diabetic retinopathy and other diabetic conditions . This is the first one to show that the eye, under normal conditions, contains endogenous, distally spliced variants of VEGF that would act to inhibit vessel growth. From the data described above, it appears that the levels of anti-angiogenic VEGF are unchanged in patients with diabetic retinopathy compared with non-diabetic patients. In the former, however, pro-angiogenic VEGF levels are preferentially up-regulated and overwhelm the anti-angiogenic family of isoforms. We do not know whether this switch is due to the diabetes or to the diabetic retinopathy. This raises many interesting scenarios for treatment and necessitates re-examination of the mechanism underlying diabetic proliferative retinopathy.
The mechanisms that underlie the control of splicing of VEGF isoforms from anti- to pro-angiogenic are completely unknown. However, it appears that in diabetes the two families of isoforms are not equally affected, suggesting that the regulation of the expression of the anti-angiogenic isoforms is different for that of the pro-angiogenic isoforms. Although some VEGF gene polymorphisms have been associated with diabetes, none of these have been linked to exon 8b splicing. None of the known polymorphisms fall in either the exon 8b region (presumably containing exon-splicing enhancers) or the intronic polypyrimidine tract upstream of exon 8b. However, regulation of splicing originating from upstream regions of the gene could result in alterations in the recruitment of splicing factors to the RNA polymerase complex and control of the speed of the polymerase reaction. It is possible, therefore, that some of the upstream polymorphisms (e.g. −460C) could affect splicing.
An understanding of the mechanism of splicing regulation may provide a method for switching off angiogenesis in the diabetic eye: if the switch can be turned off, then the increase in VEGF would be just as inhibitory as it is stimulatory for angiogenesis. Furthermore, the effects of inhibitors of VEGF on the anti-angiogenic isoforms are unknown. There have been no studies to determine whether VEGF-sequestering agents, such as VEGF-TRAP or aptamers  against VEGF, can affect pro- and anti-angiogenic isoforms equally. Specific inhibition of pro-angiogenic isoforms might appear to be a more effective strategy for preventing proliferation. With the recent findings that inhibition of VEGF proves an extremely effective treatment in phase III clinical trials  in age-related macular degeneration, it is not unreasonable to speculate that increasing the concentration of VEGFxxxb isoforms to equal or surpass those of the pro-angiogenic isoforms may be an effective therapeutic strategy for inhibiting the progression of proliferative diabetic retinopathy. It is clear that the role of VEGF splicing in the regulation of angiogenesis in the eye, and in other conditions, is going to be a key factor in our understanding of the progression of diabetic eye disease.
This work was supported by Diabetes UK (RD02/002520), the Wellcome Trust (57936, 69029), the British Heart Foundation (FS02/053 and BB2000030), the Showering Fund, the Bristol Urological Institute and the Richard Bright VEGF Research Trust.