Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life.DOI: http://dx.doi.org/10.7554/eLife.00324.001

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BackgroundAngiogenesis is a key process in several ocular disorders and cancers. Soluble Flt-1 is an alternatively spliced form of the Flt-1 gene that retains the ligand-binding domain, but lacks the membrane-spanning and intracellular kinase domains of the full-length membrane bound Flt-1 (mbFlt-1) protein. Thus, sFlt-1 is an endogenous inhibitor of VEGF-A mediated angiogenesis. Synthetic mopholino oligomers directed against splice site targets can modulate splice variant expression. We hypothesize that morpholino-induced upregulation of sFlt-1 will suppress angiogenesis in clinically relevant models of macular degeneration and breast cancer.Methods and Findings In vivo morpholino constructs were designed to target murine exon/intron 13 junction of the Flt-1 transcript denoted VEGFR1_MOe13; standard nonspecific morpholino was used as control. After nucleofection of endothelial and breast adenocarcinoma cell lines, total RNA was extracted and real-time RT-PCR performed for sFlt-1 and mbFlt-1. Intravitreal injections of VEGFR1_MOe13 or control were done in a model of laser-induced choroidal neovascularization and intratumoral injections were performed in MBA-MD-231 xenografts in nude mice. VEGFR1_MOe13 elevated sFlt-1 mRNA expression and suppressed mbFlt-1 mRNA expression in vitro in multiple cellular backgrounds (p<0.001). VEGFR1_MOe13 also elevated sFlt/mbFlt-1 ratio in vivo after laser choroidal injury 5.5 fold (p<0.001) and suppressed laser-induced CNV by 50% (p = 0.0179). This latter effect was reversed by RNAi of sFlt-1, confirming specificity of morpholino activity through up-regulation of sFlt-1. In the xenograft model, VEGFR1_MOe13 regressed tumor volume by 88.9%, increased sFlt-1 mRNA expression, and reduced vascular density by 50% relative to control morpholino treatment (p<0.05).ConclusionsMorpholino oligomers targeting the VEGFR1 mRNA exon/intron 13 junction promote production of soluble FLT-1 over membrane bound FLT-1, resulting in suppression of lesional volume in laser induced CNV and breast adenocarcinoma. Thus, morpholino manipulation of alternative splicing offers translational potential for therapy of angiogenic disorders.

The KDR gene, which participates in angiogenesis and lymphangiogenesis, produces two functionally distinct protein products, membrane-bound KDR (mbKDR) and its isoform, soluble KDR (sKDR). Since sKDR does not have a tyrosine kinase domain and does not dimerize, it is principally an antagonist of lymphangiogenesis by sequestering VEGF-C. Alternative polyadenylation of exon 30 or intron 13 leads to the production of mbKDR or sKDR, respectively, yet the regulatory mechanisms are unknown. Here we show that an antisense morpholino oligomer directed against the exon 13-intron 13 junction increases sKDR (suppressing lymphangiogenesis) and decreases mbKDR (inhibiting hemangiogenesis). The latent polyadenylation site in intron 13 of KDR is activated by blocking the upstream 5' splicing site with an antisense morpholino oligomer. Intravitreal morpholino injection suppressed laser choroidal neovascularization while increasing sKDR. In the mouse cornea, subconjunctival injection of the morpholino-inhibited corneal angiogenesis and lymphangiogenesis, and suppressed graft rejection after transplantation. Thus, this morpholino can be used for concurrent suppression of hemangiogenesis and lymphangiogenesis. This study offers new insight into the mechanisms and potential therapeutic modulation of alternative polyadenylation.