Optical transparency in the cornea and retinal photoreceptor layer depends on strict vascular demarcation. This physiologic vascular zoning is compromised in corneal injury and macular degeneration. This project will elucidate the molecular signals, mediators and regulators that sustain ocular vascular compartmentalization, by focusing on the endogenous anti-angiogenic molecule, soluble VEGFR-1 (also known as sFlt-1), and its nuclear up-regulator protein Raver2, in the cornea and the subretinal space. We previously demonstrated that corneal avascularity is dependent on soluble VEGFR-1 (sFlt-1). Morpholinos (synthetic morpholine-based oligonucleotides) upregulating sFlt-1 suppressed suture-induced corneal neovascularization and laser-induced choroidal neovascularization, and improve cornea transplant survival. In exciting new studies, we have discovered that Raver2, an endogenous nuclear regulatory protein, shifts Flt-1 splicing towards the soluble sFlt-1 isoform by modulating RNA processing. This is the first described function of Raver2 and the first identified nuclear promoter of sFlt-1 production. In the retina, we found that sFlt-1 is strongly expressed by the retinal pigment epithelium (RPE) and is vital for photoreceptor avascular privilege. We will build on these findings to characterize the regulation of sFlt-1 and Raver2 in the cornea and the subretinal space. Our central hypotheses are that sFlt-1 is vital to photoreceptor avascular privilege and that Raver2 knockdown will compromise ocular vascular demarcations.
Our specific aims are: 1. To determine the molecular regulators of sFlt-1 expression in the cornea and RPE. We will identify the nuclear factors that regulate sFlt-1 expression, validate their relevance in angiogenic assays in vivo and in vitro, and identify their RNA binding sequences. We will determine the mechanism of action of Raver2 and whether Pax6, a master regulator of ocular development, binds the Raver2 promoter. 2. To test the prediction that upregulation of sFlt-1 by Raver2 can improve corneal transplant survival and restore photoreceptor avascular privilege in models of AMD We will determine whether Raver2 can improve corneal transplant survival (by reducing corneal neovascularization and associated immune infiltration). Next, we will determine whether Raver2 can increase retinal sFlt-1 and inhibit laser induced choroidal neovascularization. 3. To determine whether genetic ablation of Raver2 induces corneal or choroidal neovascularization. Using available targeted conditional ES cells (Raver2tm1e(KOMP)Wtsi), we will develop a Raver2 knockout mouse (or Raver2-lox/loxp animal if the knockout is embryonically lethal). We will assess the impact of Raver2 knockout on development of corneal avascularity and retinal vascular demarcations pre- and post-natally, determine the presence of truncated transcripts and expression kinetics of Raver2 in the knockout, and whether Raver2 knockout affects expression of anti- or pro-angiogenic factors.

Public Health Relevance

Understanding the molecular basis of physiologic vascular zoning in the eye will yield insights into the mechanistic disruptions that lead to corneal and subretinal neovascularization, which account for several leading causes of blindness. This project will define the molecular mediators which control the regulation of sFlt-1 in the cornea and the subretinal space (focusing on Raver2, the first nuclear upregulator of sFlt-1 alternative splicing), establishing a cornerstone of understanding the development of vascular zoning in normal and pathological situations.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (DPVS)
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Mckie, George Ann
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University of Utah
Schools of Medicine
Salt Lake City
United States
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Cahoon, Judd M; Olson, Paul R; Nielson, Spencer et al. (2014) Acridine orange leukocyte fluorography in mice. Exp Eye Res 120:15-9
Das, Subrata K; Gupta, Isha; Cho, Yang Kyung et al. (2014) Vimentin knockdown decreases corneal opacity. Invest Ophthalmol Vis Sci 55:4030-40
Chennamaneni, Srinivas Rao; Mamalis, Christina; Archer, Bonnie et al. (2013) Development of a novel bioerodible dexamethasone implant for uveitis and postoperative cataract inflammation. J Control Release 167:53-9
Singh, Nirbhai; Tiem, Michelle; Watkins, Ryan et al. (2013) Soluble vascular endothelial growth factor receptor 3 is essential for corneal alymphaticity. Blood 121:4242-9
Uehara, Hironori; Cho, YangKyung; Simonis, Jackie et al. (2013) Dual suppression of hemangiogenesis and lymphangiogenesis by splice-shifting morpholinos targeting vascular endothelial growth factor receptor 2 (KDR). FASEB J 27:76-85
Cho, Yang Kyung; Uehara, Hironori; Young, Jason R et al. (2012) Vascular endothelial growth factor receptor 1 morpholino decreases angiogenesis in a murine corneal suture model. Invest Ophthalmol Vis Sci 53:685-92
Qazi, Yureeda; Stagg, Brian; Singh, Nirbhai et al. (2012) Nanoparticle-mediated delivery of shRNA.VEGF-a plasmids regresses corneal neovascularization. Invest Ophthalmol Vis Sci 53:2837-44
Owen, Leah A; Uehara, Hironori; Cahoon, Judd et al. (2012) Morpholino-mediated increase in soluble Flt-1 expression results in decreased ocular and tumor neovascularization. PLoS One 7:e33576
Kleinman, Mark E; Kaneko, Hiroki; Cho, Won Gil et al. (2012) Short-interfering RNAs induce retinal degeneration via TLR3 and IRF3. Mol Ther 20:101-8
Tarallo, Valeria; Hirano, Yoshio; Gelfand, Bradley D et al. (2012) DICER1 loss and Alu RNA induce age-related macular degeneration via the NLRP3 inflammasome and MyD88. Cell 149:847-59

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