Functional significance of VEGF regulation by SRPK1

Human beings have remarkably few genes - about 22000 in total. Despite this we can make hundreds of thousands of different gene products - the proteins that make the body work. Each gene can generate multiple proteins by use of different parts of the gene - a process called alternative splicing. In this process different sequences coded by the DNA from the gene are stuck together to give a different message to make proteins. This is analogous to splicing together different scenes from a film to make different stories - cutting in a different ending can change a story from a tragedy to a romance. One of the most important processes in human development, normal function and disease, is how blood vessels grow and respond to injury - including in cancer, diabetes, blindness and kidney disease. Blood vessels require a balance between two types of the growth factor VEGF to work properly. These two types are made by this alternative splicing process. One type of VEGF makes new blood vessels that are leaky, grow into new tissue, and contribute to the most common forms of blindness (wet age related macular degeneration and diabetic retinopathy), kidney failure and cancer. The other type, made by splicing in a different ending to the protein, prevents new vessel growth and protects kidneys from failure in diabetes, and blindness and cancer in animals. The editing process that the cell uses to decide which of these two forms are generated has a number of steps in it, some of which provide specificity, to allow different forms of molecules to be made. We previously found that these steps are carried out by two proteins, SRPK1 and SRSF1. This project will find out how these molecules work, and whether inhibiting them can prevent blindness and hereditary kidney disease in animals. We will do this by using newly discovered drugs and genetic mouse models that have been developed to study these molecules, and by generating a new mouse model that will help us understand how SRPK1 works.

Altered blood vessel growth and permeability by altered VEGF-A signalling underpin many diseases of the western world. VEGF-A is generated as two different isoforms families - pro-angiogenic, pro-permeability VEGF-A165a, and anti-angiogenic, anti-permeabiity, cytoprotective VEGF-A165b - by alternative splicing. We recently identified splice factors that regulate this alternative splicing, including the protein kinase SRPK1, which acts by phosphorylating SRSF1 in kidney and retinal epithelial cells to switch splicing from VEGF165b to VEGF165a. Mutations in WT1 that result in the proteinuric kidney disease Denys Drash Syndrome have increased SRPK1 expression in the kidney, and this prevents splicing to VEGF-A165b. Moreover, small molecular weight inhibitors of SRPK1 can prevent angiogenesis in the eye. Thus SRPK1 appears to be linked to disorders of both permeability (glomerular permeability results in proteinuria), and angiogenesis. However, the quantitative and qualitative contribution of SRPK1 to VEGF mediated permeability and angiogenesis is not known. We will define this contribution using minigenes that we have recently generated that can track the splicing events in cells in culture. We also intend to investigate the contribution of SRPK1 in controlling VEGF splicing in health and disease by generating tissue specific knockout and over-expressing mice in which SRPK1 expression is inhibited or activated. We will bring in LoxP-SRPK1 mice from Xian-Dong Fu at University of California at San Diego and generate a TetO-SRPK1 mouse. We will cross our existing inducible kidney specific (Pod-rtTA/TetO-Cre) and eye specific (MCT-3-Cre from Steve Moss, UCL) mouse lines with these lines to result in podocyte and eye specific inducible knockout and over-expressing mice, which will be used for angiogenesis assays and glomerular permeability assays and cross the podocyte specific mice with WT1 mutant mice to determine the role of SRPK1 in Denys Drash Syndrome.

On-going improvements in life expectancy have reinforced the need for robust therapies for the major killers in the developed world - vascular disease, malignancy and the complications of diabetes. Indeed the DOH Confidential Mortality statistics reveals that over 80% of UK deaths result from these conditions, conditions that consume large amounts of NHS resources. Of note, the pathophysiology of all these conditions is characterised by abnormal proliferation and/or permeability of the smallest vessels in the body - the Microvessels. The biology of micro-vessels is profoundly influenced by two opposing families of isoforms of Vascular Endothelial Growth Factor-A (VEGF-A). One family is commonly associated with disease promoting microvessel proliferation and hyper-permeability, the other family beneficially opposes these effects. Crucially these contrasting families derive from the same gene. Our highly specialised resources and skills is unrivalled worldwide and will provide a unique opportunity to elucidate how the VEGF-isoform family balance may be orchestrated and controlled. This project may therefore allow the development of novel strategies to maintain or re-establish normal micro-vessel function (Estimated timescale 5-10 yrs). Our findings may therefore have profound implications, not only for patients and those at risk of cardiovascular disease, cancer and diabetes, but also for the financial drain on health service providers (Estimated Timescale 10-15yrs). These diseases are so prevalent that even modest improvements in their prevalence or progression would have profound beneficial implications for the mortality of these individuals and also on health economics and the regulators of resource re-allocation.
The Microvascular Research Laboratories' ethos is one of maximizing translation and we have ensured the Intellectual property is in place surrounding VEGF to allow inward investment from the commercial private sector and licensing to industry for any agents that can influence the all important balance between VEGF isoform families.
Not only do we strive for a translational benefit to patients mortality directly as above, but also indirectly since these conditions (as well as others characterised by microvessel dysfunction eg Rheumatoid Disease, Chronic Chest disease), not only shorten life expectancy, but are often characterised by years or decades of pain, immobility, dependence, disability, reduced self esteem and social isolation. It is hoped therefore that indirect benefits would also ensue to patients, carers, social networks, charitable NGOs, and the wider society by the minimisation of the morbidity associated with these patterns of disease improving quality of life, reducing dependence, increasing employments prospects etc.