Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC231
SRPK1 inhibition significantly reduces laser-induced choroidal neovascularisation (CNV) and intravitreal neovascularisation (IVNV) in an Oxygen Induced Retinopathy (OIR) model of Retinopathy of Prematurity (ROP) in rats
M. V. Gammons1, D. Bates1
1. University of Bristol, Bristol, United Kingdom.
Vascular endothelial growth factor (VEGF) induces abnormal growth of retinal blood vessels in Retinopathy of Prematurity (ROP), a leading cause of blindness in children. Serine rich protein kinase 1 (SRPK1) has been identified as a target in controlling the splicing of VEGF(1). The VEGF gene is alternatively spliced into two sister families of isoforms, angiogenic VEGFxxx and anti-angiogenic, VEGFxxxb. Both VEGFxxx and VEGFxxxb isoforms are expressed in the normal retina. SRPK1 phosphorylates and induces nuclear localization of alternative splice factor/splice factor 2 (ASF/SF2), a promoter of VEGFxxx splice site selection, thus upregulating pro-angiogenic VEGF production. We investigated whether an inhibitor of SRPK1/2, SRPIN340(2) could alter VEGF splicing to promote VEGFxxxb isoforms and inhibit pathological neovascularisation in models of eye disease. Newborn rat pups to repeated cycles of 24 h of 50% oxygen alternating with 24h of 10% oxygen (50/10 OIR model) to cause a condition similar to human ROP. At post-natal day (P) 12 pups were anaesthetized by intraperitoneal (IP) injection of medetomidine (0.5mg/kg) and ketamine (50mg/kg), and given a 2μl intraocular (IO) injection of 10ng/μl SRPIN340 or saline. At P14 pups were removed from the oxygen chamber and left in normoxia until P20, when they were killed and the retinas removed, and flat-mounted. Additionally SRPIN340 was tested in a laser induced CNV model in Norway Brown rats, rats were anaesthetized by an IP injection of medetomidine (0.5mg/kg) and ketamine (50mg/kg). Following laser photocoagulation rats received IO injections of 25ng SRPIN340 in the ipsilateral eye and saline in the contralateral eye. 7 days later the rats were anaesthetized as above, and the injections repeated. 14 days after laser photocoagulation animals were killed by inhalation of isofluorane, and choroids dissected and flat mounted. Retinas or choroids were isolectin stained, and IVNV or CNV was determined by area of pathology (ImageJ). SRPIN340 (p<0.05, students paired t-test) significantly reduced the IVNV area from 1.04±0.15% of the retina in control eyes (p>0.05) to 0.74±0.13% of total retina and significantly reduced CNV area (p<0.05, students paired t-test) in treated eyes (9916±2159µm2) compared to matched controls (18027±1732µm2). These results indicate that targeting SRPK1, which has been shown to switch splicing from pro to anti-angiogenic splice variants of VEGF, has the potential to prevent VEGF mediated pathological blood vessel growth in vivo. This approach would maintain the production of cytoprotective VEGFxxxb isoforms compared with current anti-VEGF therapeutics that target all VEGF isoforms.
Where applicable, experiments conform with Society ethical requirements