Hideyuki Onami

Micropatterned Polymeric Nanosheets for Local Delivery of an Engineered Epithelial Monolayer

Like a carpet for cells, micropatterned polymeric nanosheets are developed toward local cell delivery. The nanosheets direct morphogenesis of retinal pigment epithelial (RPE) cells and allow for the injection of an engineered RPE monolayer through syringe needles without the loss of cell viability. Such an ultrathin carrier has the promise of a minimally invasive delivery of cells into narrow tissue spaces.

A scalable controlled-release device for transscleral drug delivery to the retina.

A transscleral drug-delivery device, designed for the administration of protein-type drugs, that consists of a drug reservoir covered with a controlled-release membrane was manufactured and tested. The controlled-release membrane is made of photopolymerized polyethylene glycol dimethacrylate (PEGDM) that contains interconnected collagen microparticles (COLs), which are the routes for drug permeation. The results showed that the release of 40-kDa FITC-dextran (FD40) was dependent on the COL concentration, which indicated that FD40 travelled through the membrane-embedded COLs. Additionally, the sustained-release drug formulations, FD40-loaded COLs and FD40-loaded COLs pelletized with PEGDM, fine-tuned the release of FD40. Capsules filled with COLs that contained recombinant human brain-derived neurotrophic factor (rhBDNF) released bioactive rhBDNF in a manner dependent on the membrane COL concentration, as was found for FD40 release. When capsules were sutured onto sclerae of rabbit eyes, FD40 was found to spread to the retinal pigment epithelium. Implantation of the device was easy, and it did not damage the eye tissues. In conclusion, our capsule is easily modified to accommodate different release rates for protein-type drugs by altering the membrane COL composition and/or drug formulation and can be implanted and removed with minor surgery. The device thus has great potential as a conduit for continuous, controlled drug release.

Transscleral Sustained Vasohibin-1 Delivery by a Novel Device Suppressed Experimentally-Induced Choroidal Neovascularization

We established a sustained vasohibin-1 (a 42-kDa protein), delivery device by a novel method using photopolymerization of a mixture of polyethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and collagen microparticles. We evaluated its effects in a model of rat laser-induced choroidal neovascularization (CNV) using a transscleral approach. We used variable concentrations of vasohibin-1 in the devices, and used an enzyme-linked immunosorbent assay and Western blotting to measure the released vasohibin-1 (0.31 nM/day when using the 10 μM vasohibin-1 delivery device [10VDD]). The released vasohibin-1 showed suppression activity comparable to native effects when evaluated using endothelial tube formation. We also used pelletized vasohibin-1 and fluorescein isothiocyanate-labeled 40 kDa dextran as controls. Strong fluorescein staining was observed on the sclera when the device was used for drug delivery, whereas pellet use produced strong staining in the conjunctiva and surrounding tissue, but not on the sclera. Vasohibin-1 was found in the sclera, choroid, retinal pigment epithelium (RPE), and neural retina after device implantation. Stronger immunoreactivity at the RPE and ganglion cell layers was observed than in other retinal regions. Significantly lower fluorescein angiography (FA) scores and smaller CNV areas in the flat mounts of RPE-choroid-sclera were observed for the 10VDD, VDD (1 μM vasohibin-1 delivery device), and vasohibin-1 intravitreal direct injection (0.24 μM) groups when compared to the pellet, non-vasohibin-1 delivery device, and intravitreal vehicle injection groups. Choroidal neovascularization can be treated with transscleral sustained protein delivery using our novel device. We offer a safer sustained protein release for treatment of retinal disease using the transscleral approach.

Suppression of Choroidal Neovascularization by Vasohibin-1, a Vascular Endothelium–Derived Angiogenic Inhibitor

PURPOSE. To determine the expression of vasohibin-1 during the development of experimentally induced choroidal neovascularization (CNV) and to investigate the effect of vasohibin-1 on the generation of CNV. METHODS. CNV lesions were induced in the eyes of wild-type (WT) and vasohibin-1 knockout (KO) mice by laser photocoagulation. The expression of vasohibin-1, vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR1), VEGFR2, and pigment epithelial-derived factor (PEDF) was determined by semiquantitative reverse transcription-polymerase chain reaction. The expression of vasohibin-1 was also examined by immunohistochemistry with anti-CD68, anti-alpha smooth muscle actin (αSMA), anti-cytokeratin, and anti-CD31. Vasohibin-1 was injected into the vitreous and the activity and size of the CNV were determined by fluorescein angiography and in choroidal flat mounts. RESULTS. Vasohibin-1 was detected not only in CD31-positive endothelial cells but also in CD68-positive macrophages and αSMA-positive retinal pigment epithelial cells. Strong vasohibin-1 expression was observed at day 28, when the CNV lesions had regressed by histologic examination. The vasohibin-1 level was significantly decreased at day 14 and increased at day 28 after laser application. Significantly less VEGFR2 expression was observed on day 4 after vasohibin-1. The expression of PEDF was not significantly changed by vasohibin-1 injection. Vasohibin-1 injection significantly suppressed the CNV, with no adverse side effects. The CNV lesions in the vasohibin-1-KO mice were significantly larger than those in the WT mice. CONCLUSIONS. The endogenous expression of vasohibin-1 is associated with the natural course of the development of CNV. Intravitreal injections of vasohibin-1 may be a method for inhibiting CNV.

A Platform for Controlled Dual-Drug Delivery to the Retina: Protective Effects against Light-Induced Retinal Damage in Rats

Controlled transscleral co-delivery of two drugs, edaravone (EDV) and unoprostone (UNO), using a platform that comprises a microfabricated reservoir, controlled-release cover, and drug formulations, which are made of photopolymerized poly(ethyleneglycol) dimethacrylates, shows synergistic retinal neuroprotection against light injury in rats when compared with single-drug-loaded devices. The device would offer a safer therapeutic method than intravitreal injections for retinal disease treatments.

Reduction of laser-induced choroidal neovascularization by intravitreal vasohibin-1 in monkey eyes.

Purpose: To determine whether intravitreal vasohibin-1 will reduce the grade of the choroidal neovascularization in monkey eyes. Methods: Choroidal neovascularizations were induced in 12 monkey eyes by laser photocoagulation. Three monkeys were evaluated for the safety of the vasohibin-1 injections, 6 monkeys for the effects of a single injection, and 3 monkeys for repeated injections of vasohibin-1. Ophthalmoscopy, fluorescein angiography, focal electroretinograms, and optical coherence tomography were used for the evaluations. The level of vascular endothelial growth factor in the aqueous was determined by enzyme-linked immunosorbent assay. Immunohistochemistry was performed. Results: An intravitreal injection of 10 &mgr;g of vasohibin-1 induced mild intraocular inflammation. Eyes with an intravitreal injection of 0.1 &mgr;g and 1.0 &mgr;g of vasohibin-1 had significant less fluorescein leakage from the choroidal neovascularizations and larger amplitude focal electroretinograms than that of vehicle-injected eyes. Similar results were obtained by repeated injections of 0.1 &mgr;g of vasohibin-1. Immunohistochemistry showed that vasohibin-1 was expressed mainly in the endothelial cells within the choroidal neovascularizations. The vascular endothelial growth factor level was not significantly altered by intravitreal vasohibin-1. Conclusion: The reduction of the laser-induced choroidal neovascularizations and preservation of macular function in monkey by intravitreal vasohibin-1 suggest that it should be considered for suppressing choroidal neovascularizations in humans.

Protective effects of sustained transscleral unoprostone delivery against retinal degeneration in S334ter rhodopsin mutant rats.

It has been suggested that unoprostone isopropyl (UNO) has potent neuroprotective activity in the retina. The effect of sustained transscleral UNO delivery to the posterior segment of the eye on photoreceptor degeneration was evaluated. UNO was loaded into a device made of poly(ethyleneglycol) dimethacrylate by polydimethylsiloxane mold-based UV-curing. The amount of UNO diffusing from these devices was measured using high-performance liquid chromatography. The polymeric devices that released UNO at 1.8 μg/day were implanted on the sclerae of S334ter rats at postnatal 21 days, and electroretinograms (ERGs) were compared with those of topical application and placebo devices. Retinal thickness was evaluated by histological examination. Western blots of specimens 4 weeks after implantation were performed. ERGs showed that the UNO-loaded device prevented the reduction of ERG amplitudes 2 and 4 weeks after implantation, compared with results using a placebo device or topical application. Histological examination showed that the UNO-loaded device prevented the reduction of retinal thickness, and Western blots of specimens indicated that the UNO-loaded device decreased expression of ERK1/2, phosphorylated ERK1/2, and caspase-3. A device that provided sustained UNO administration protected against retinal degeneration in rhodopsin mutant rats, and thus, may have translational potential as a sustainable method to administer drugs to treat retinitis pigmentosa.

Intrascleral Transplantation of a Collagen Sheet with Cultured Brain-Derived Neurotrophic Factor Expressing Cells Partially Rescues the Retina from Damage due to Acute High Intraocular Pressure

We constructed brain-derived neurotrophic factor (BDNF) expressing rat retinal pigment epithelial (RPE) cells by stable transfection of BDNF cDNA, and the RPE cells were cultured on a cross-linked collagen sheet (Coll-RPE-BDNF). BDNF expression of the Coll-RPE-BDNF was confirmed by western blot, and the Coll-RPE-BDNF was transplanted into the rabbit sclera. In vivo BDNF expression was confirmed by His expression that was linked to the expressing BDNF. The effect of the released BDNF was examined in a rabbit acute high intraocular pressure system by electroretinogram and histological examination. Statistically significant preservation of ERG b wave amplitude was observed in the rabbits treated by Coll-RPE-BDNF when compared to that of no treatment. Statistically significant preservation of the thickness of the inner nuclear layer at the transplanted area was observed in the rabbits treated by Coll-RPE-BDNF compared to that of no treatment. Intra-scleral Coll-RPE-BDNF transplantation may partially rescue retinal cells from acute high intraocular pressure.

Vasohibin-1 and Retinal Pigment Epithelium

Vascular endothelial growth factor (VEGF) is one of the main factors for inducing choroidal neovascularization in patients with age-related macular degeneration (AMD). Retinal pigment epithelium (RPE) is one of the main sources for expressing VEGF. Vasohibin-1 is a VEGF-inducible gene in human cultured endothelial cells with antiangiogenic properties. We examined the effects of vasohibin-1 against RPE. We used rat RPE cell line, RPE-J. Cobalt chloride and low glucose and oxygen supply were used for hypoxic stress. Western blot analysis and real-time PCR were performed to detect the expression of vasohibin-1 and VEGF in the RPE-J. Cobalt chloride or low oxygen and low glucose enhanced VEGF expression, whereas statistically significant less vasohibin-1 expression was observed. RPE cell dynamics was monitored using xCELLigence System for real-time cell analysis during culture. External VEGF (0.2–2 nM) enhanced Cell Index (CI), such as cell viability, number, and adhesion to the plates significantly at 2% oxygen and no glucose when compared to those of vehicle or other concentration of VEGF. Conversely, external vasohibin-1 (2 nM) showed lower CI at the indicated condition. Vasohibin-1 also reduced VEGF-induced CI index. These results were not observed under standard culture condition. When we performed MTS assay or cell count, external vasohibin-1 also showed comparable results. When we transduced full-length vasohibin-1 cDNA in RPE-J, the internal vasohibin-1 showed less VEGF expression than that of control vector-transduced RPE-J. In summary, vasohibin-1 showed opposite results as that of VEGF on the RPE-J, especially under hypoxic condition.