Jacquelyn Simonis

Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1

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

Nanoparticle-Mediated Delivery of shRNA.VEGF-A Plasmids Regresses Corneal Neovascularization

PURPOSE To determine the efficacy of a plasmid containing a small hairpin RNA expression cassette (pSEC.shRNA) against VEGF-A-loaded poly(lactic co-glycolic acid) nanoparticles (PLGA NPs) in the sustained regression of murine corneal neovascularization. METHODS PLGA nanoparticles were loaded with pSEC.shRNA.VEGF-A plasmids using the double emulsion-solvent evaporation method. KNV was induced in BALB/c mice by mechanical-alkali injury. Four weeks after induction of KNV, the mice were randomly divided to receive one of four treatments intrastromally: pSEC.shRNA.VEGF-A PLGA NPs (2 μg plasmid); naked pSEC.shRNA.VEGF-A plasmid only (2 μg plasmid); control blank PLGA NPs (equivalent dry weight of NPs); and vehicle. Two and five days after intervention, corneas were harvested to determine VEGF-A gene and protein expression using reverse transcriptase polymerase chain reaction and ELISA, respectively. Four weeks after intervention, corneas were photographed, mice sacrificed, and the corneal whole mounts were immunostained for CD31 (panendothelial cell marker). Immunofluorescence microscopy was performed and the neovascular area was quantitated. RESULTS VEGF-A mRNA (49.6 ± 12.4 vs. 82.9 ± 6.0%, P < 0.01) and protein (4.0 ± 5.2 vs. 20.0 ± 7.5 ρg VEGF-A/mg total protein, P < 0.05) expression were significantly reduced in pSEC.shRNA.VEGF-A PLGA NP-treated corneas as compared with control blank NP. The pSEC.shRNA.VEGF-A PLGA NP-treated corneas showed significant regression in the mean fractional areas of KNV (0.125 ± 0.042; 12.5%, P <0.01) compared with both naked plasmid only (0.283 ± 0.004; 28.3%) and control (blank NPs = 0.555 ± 0.072, 55.5%) at 4 weeks post-treatment. CONCLUSIONS The pSEC.shRNA.VEGF-A-loaded PLGA NPs are an effective, nonviral, nontoxic, and sustainable form of gene therapy for the regression of murine KNV.

Morpholino-mediated increase in soluble Flt-1 expression results in decreased ocular and tumor neovascularization

Background Angiogenesis 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). Conclusions Morpholino 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 capsule drug device: Novel approach for drug delivery to the eye

Treatment of age-macular degeneration requires monthly intravitreal injections, which are costly and have serious risks. The objective of this study was to develop a novel intraocular implant for drug delivery. The capsule drug ring is a reservoir inserted in the lens capsule during cataract surgery, refillable and capable of delivering multiple drugs. Avastin was the drug of interest in this study. Prototypes were manufactured using polymethylmethacrylate sheets as the reservoir material, a semi-permeable membrane for controlled delivery and silicone check valves for refilling. The device showed near zero-order release kinetics and Avastin stability was investigated with accelerated temperature studies.

Anti-SPARC oligopeptide inhibits laser-induced CNV in mice

It is known that SPARC gates VEGF-A signal transduction towards KDR, the primary angiogenic VEGF receptor. We sought to determine whether inhibition of SPARC activity using anti-SPARC peptide could inhibit laser-induced CNV by promoting binding of VEGF-A to FLT-1. We created anti-SPARC l-peptide and retro-inverso anti-SPARC d-peptide. Anti-SPARC peptides or PBS were injected intravitreally 1day before or after laser induction. Intravitreal injection of anti-SPARC l-peptide 1day before laser induction promotes FLT-1 phosphorylation and inhibited laser-induced CNV and anti-SPARC d-peptide had no effect. Injection 1day after laser injury did not affect size of laser-induced CNV. Inhibition of SPARC activity could be complementary to existing anti-CNV therapy.