Eiji Sakurai

Sequence- and target-independent angiogenesis suppression by siRNA via TLR3

Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-α/β activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-γ and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3–RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world’s population, and that siRNAs might induce unanticipated vascular or immune effects.

An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice

The study and treatment of age-related macular degeneration (AMD), a leading cause of blindness, has been hampered by a lack of animal models. Here we report that mice deficient either in monocyte chemoattractant protein-1 (Ccl-2; also known as MCP-1) or its cognate C-C chemokine receptor-2 (Ccr-2) develop cardinal features of AMD, including accumulation of lipofuscin in and drusen beneath the retinal pigmented epithelium (RPE), photoreceptor atrophy and choroidal neovascularization (CNV). Complement and IgG deposition in RPE and choroid accompanies senescence in this model, as in human AMD. RPE or choroidal endothelial production of Ccl-2 induced by complement C5a and IgG may mediate choroidal macrophage infiltration into aged wild-type choroids. Wild-type choroidal macrophages degrade C5 and IgG in eye sections of Ccl2−/− or Ccr2−/− mice. Impaired macrophage recruitment may allow accumulation of C5a and IgG, which induces vascular endothelial growth factor (VEGF) production by RPE, possibly mediating development of CNV. These models implicate macrophage dysfunction in AMD pathogenesis and may be useful as a platform for validating therapies.

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity—the absence of blood vessels in the cornea—is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

Effect of Particle Size of Polymeric Nanospheres on Intravitreal Kinetics

In this study, we injected nanospheres containing a fluorescein derivative into the vitreous cavity of pigmented rabbit eyes and evaluated their intraocular kinetics as drug carriers in vivo. Polystyrene nanospheres (2 µm, 200 nm and 50 nm in diameter) containing a fluorescein derivative were used in this study. A suspension of each particle was prepared by diluting with distilled water at a concentration of 10 µg/ml equivalent to sodium fluorescein. The suspension of nanospheres was injected once into the vitreous cavity of unilateral eyes of pigmented rabbits. A sodium fluorescein solution of the same concentration was injected once into the vitreous cavity of the other eye as the control. The intraocular kinetics of nanospheres was evaluated by measuring vitreous fluorescence using a scanning fluorophotometer. To investigate elimination pathways of nanospheres in detail, serial cross-sections of the eyes were examined with a fluorescence microscope. The fluorescence derived from nanospheres was observed in the vitreous cavity for over 1 month (2 µm: t1/2 = 5.4 ± 0.8 days, 200 nm: t1/2 = 8.6 ± 0.7 days, 50 nm: t1/2 = 10.1 ± 1.8 days), whereas that in the control eyes completely disappeared within 3 days (t1/2 = 7.8 ± 0.7 h). The elimination half-life from the vitreous cavity correlated well with the particle diameter (r = –0.997, p = 0.007). Histological studies using a fluorescence microscope revealed that nanospheres with a diameter of 2 µm were seen in the vitreous cavity and trabecular meshwork, while nanospheres with a diameter of smaller than 200 nm were also observed in the retina as well as these tissues. Our findings indicated that nanospheres may be beneficial as a drug carrier to the retina, vitreous and trabecular meshwork.

Loss of SPARC-mediated VEGFR-1 suppression after injury reveals a novel antiangiogenic activity of VEGF-A

VEGF-A promotes angiogenesis in many tissues. Here we report that choroidal neovascularization (CNV) incited by injury was increased by excess VEGF-A before injury but was suppressed by VEGF-A after injury. This unorthodox antiangiogenic effect was mediated via VEGFR-1 activation and VEGFR-2 deactivation, the latter via Src homology domain 2-containing (SH2-containing) tyrosine phosphatase-1 (SHP-1). The VEGFR-1-specific ligand placental growth factor-1 (PlGF-1), but not VEGF-E, which selectively binds VEGFR-2, mimicked these responses. Excess VEGF-A increased CNV before injury because VEGFR-1 activation was silenced by secreted protein, acidic and rich in cysteine (SPARC). The transient decline of SPARC after injury revealed a temporal window in which VEGF-A signaling was routed principally through VEGFR-1. These observations indicate that therapeutic design of VEGF-A inhibition should include consideration of the level and activity of SPARC.

Retention of dye after indocyanine Green-assisted internal limiting membrane peeling ☆

PURPOSE To describe the long-term retention of indocyanine green (ICG) in the fundus after ICG-assisted internal limiting membrane peeling. DESIGN Case report. Two patients underwent vitrectomy including ICG-assisted internal limiting membrane peeling. The fundus was examined with a 780-nm infrared illumination of a scanning laser ophthalmoscope after surgery. RESULTS No ICG staining of the fundus was visible ophthalmoscopically. Examination with a scanning laser ophthalmoscope, however, detected fluorescence from residual ICG until 6 months after surgery in case 1 and 9 months in case 2. No complication related to the residual ICG was observed. CONCLUSIONS The results suggested that ICG remains in the fundus for a long period after surgery. Clearance of the dye from the diabetic retina may be prolonged.

Drug delivery from ocular implants

Developing an intraocular drug delivery system (DDS) is urgently needed because most vitreoretinal diseases are refractory to conventional pharmacological approaches; eye drops and systemically administered drugs cannot deliver therapeutic drug concentrations into vitreoretinal tissue. Intraocular DDSs address this problem. Intraocular sustained-drug release via implantable devices or injectable microparticles has been investigated to treat vitreoretinal diseases. A nonbiodegradable implant was first used in 1996 for cytomegalovirus retinitis secondary to the acquired immunodeficiency syndrome. Biodegradable implants, composed of hydrophilic or hydrophobic polymers, in the shape of rods, plugs, discs or sheets have been investigated. An injectable rod is presently being assessed in a Phase III trial to treat macular oedema secondary to diabetic retinopathy or branch-retinal vein occlusion. Intraocular DDSs using a biodegradable implant may soon be successfully used to treat serious intraocular disorders.

Ocular and systemic features of Peters' anomaly.

Abstract Background: To clarify the relationship between associated systemic anomalies and ocular manifestations in patients with Peters’ anomaly, a retrospective study was conducted. Methods: We classified 37 patients with Peters’ anomaly into two groups, one with (+) and one without (–) systemic anomalies. Results: The systemic anomaly (+) group consisted of 13 patients, eight males and five females, with mean age of 2.3 months. Peters’ anomaly was bilateral in six cases and unilateral in seven. Corneolenticular adhesion was observed in 11 cases. Associated ocular anomalies were seen in 12 cases, and developmental glaucoma was present in eight cases. The systemic anomaly (–) group comprised 24 patients, 13 males and 11 females, with mean age of 28.3 months. Peters’ anomaly was bilateral in 11 cases and unilateral in 13. Corneolenticular adhesion was observed in five cases. The associated ocular anomalies were observed in 10 cases, and developmental glaucoma was accompanied in six cases. The incidences of cases with corneolenticular adhesion, those with other ocular anomalies, and those with glaucoma were significantly higher in the systemic anomaly (+) group than in the systemic anomaly (–) group. Conclusions: Peters’ anomaly accompanying corneolenticular adhesion and/or other ocular anomalies should be evaluated for the presence of systemic anomalies.

Late-onset laser in situ keratomileusis (LASIK) flap dehiscence during retinal detachment surgery

PURPOSE To report a case of laser in situ keratomileusis (LASIK) flap dehiscence during retinal detachment surgery 7 months after uneventful refractive surgery. DESIGN Interventional case report. METHODS A 47-year-old man noticed a defect of the upper visual field in his right eye 7 months after a LASIK procedure. The fundus showed rhegmatogenous retinal detachment, and a scleral buckling procedure was performed. During the buckling procedure, the corneal flap became detached. RESULTS At completion of the buckling procedure, the detached corneal flap was carefully raised and the exposed corneal stroma was cleansed of any residual epithelial cells or red blood cells with irrigation using balanced salt solution. One day after the operation, the LASIK flap was repositioned, the cornea had cleared, and the retina was reattached. CONCLUSIONS As LASIK increases in popularity, the complication we have reported may become more common. We suggest that a retinal detachment surgery should be performed with careful avoidance of corneal trauma even if a long time has passed since the LASIK procedure.

Intraocular pressure elevation following triamcinolone acetonide administration as related to administration routes

PurposeTo evaluate the incidence and risk factors of intraocular pressure (IOP) elevation following triamcinolone acetonide (TA) administration.MethodsIn this retrospective observational case series, patients (224 eyes of 202 patients) with diffuse diabetic macular edema (66 eyes), branch retinal vein occlusion (39 eyes), central retinal vein occlusion (25 eyes), exudative age-related macular degeneration (49 eyes), myopic choroidal neovascularization (10 eyes), uveitis (30 eyes), or other conditions (5 eyes) were administered an intravitreal or posterior sub-Tenon capsule injection, or both, of TA. Sub-Tenon capsule injection was performed on 106 eyes (STTA group). Intravitreal injection was performed on 118 eyes (IVTA group), of which 85 eyes underwent simultaneous intravitreal and sub-Tenon capsule injections. Mean follow-up after TA administration was 15.9 ± 10.4 (range, 3–39) months. The sub-Tenon capsule injection and intravitreal injection of TA were compared with respect to the frequency of IOP elevation and the time between TA administration and the initial IOP elevation, and the possible risk factors responsible for IOP elevation were identified.ResultsThere was no significant difference in frequency of IOP > 21 mmHg between the STTA group and the IVTA group (P = 0.0588). There was, however, a significant difference in the frequency of IOP > 30 mmHg between the two groups (P = 0.0004). In the IVTA group, more patients needed antiglaucoma medication than in the STTA group (P = 0.0052). The incidence rate of IOP elevation within 1 week after TA administration in the IVTA group was significantly higher than in the STTA group (P = 0.0154). Risk factors for IOP elevation included higher baseline IOP (P < 0.0001), younger patients (P = 0.0095), and simultaneous administration of sub-Tenon capsule and intravitreal injections (P = 0.0228).ConclusionsCareful follow-up of IOP is required after TA injections.