RESUMEN

A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early-onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through the loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore the phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.

Asunto(s)

Sistemas CRISPR-Cas/genética , Codón Iniciador/genética , Distrofia Endotelial de Fuchs , Edición Génica/métodos , Animales , Colágeno Tipo VIII/genética , Modelos Animales de Enfermedad , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/prevención & control , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Guía de Kinetoplastida/genética

RESUMEN

Purpose: Neurotrophic keratopathy is a degenerative disease that may be improved by nerve growth factor (NGF). Our aim was to investigate the use of pergolide, a dopamine (D1 and D2) receptor agonist known to increase the synthesis and release of NGF for regeneration of damaged corneal nerve fibers. Methods: Pergolide function was evaluated by measuring axon length and NGF levels by enzyme-linked immunosorbent assay in cultured chicken dorsal root ganglion (DRG) cells with serial doses of pergolide (10, 25, 50, 150, and 300 µg/ml) and with different concentrations of a D1 antagonist. Pergolide function was further evaluated by cornea nerve fiber density and wound healing in a cornea scratch mouse model. Results: Pergolide increased DRG axon length significantly at a dose between 50 and 300 µg/ml. Different concentrations of D1 antagonist (12, 24, 48, and 96 µg/ml) inhibited DRG axon length growth with pergolide (300 µg/ml). Pergolide (50 µg/ml) upregulated NGF expression in DRG cells at both 24 hours and 48 hours. Pergolide improved cornea nerve fiber density at both 1 week and 2 weeks. Pergolide also improved cornea wound healing. Conclusions: We demonstrated that pergolide can act to promote an increase in NGF which promotes corneal nerve regeneration and would therefore improve corneal sensation and visual acuity in eyes with peripheral neurotrophic keratopathy.

Asunto(s)

Lesiones de la Cornea/tratamiento farmacológico , Agonistas de Dopamina/uso terapéutico , Fibras Nerviosas/efectos de los fármacos , Pergolida/uso terapéutico , Animales , Axones/efectos de los fármacos , Pollos , Agonistas de Dopamina/farmacología , Ganglios Espinales/efectos de los fármacos , Ratones , Regeneración Nerviosa , Pergolida/farmacología , Cicatrización de Heridas/fisiología

RESUMEN

Purpose: We determine whether intravitreal angiopoietin-1 combined with the short coiled-coil domain of cartilage oligomeric matrix protein by adeno-associated viral serotype 2 (AAV2.COMP-Ang1) delivery following the onset of vascular damage could rescue or repair damaged vascular beds and attenuate neuronal atrophy and dysfunction in the retinas of aged diabetic mice. Methods: AAV2.COMP-Ang1 was bilaterally injected into the vitreous of 6-month-old male Ins2Akita mice. Age-matched controls consisted of uninjected C57BL/6J and Ins2Akita males, and of Ins2Akita males injected with PBS or AAV2.REPORTER (AcGFP or LacZ). Retinal thickness and visual acuity were measured in vivo at baseline and at the 10.5-month endpoint. Ex vivo vascular parameters were measured from retinal flat mounts, and Western blot was used to detect protein expression. Results: All three Ins2Akita control groups showed significantly increased deep vascular density at 10.5 months compared to uninjected C57BL/6J retinas (as measured by vessel area, length, lacunarity, and number of junctions). In contrast, deep microvascular density of Ins2Akita retinas treated with AAV2.COMP-Ang1 was more similar to uninjected C57BL/6J retinas for all parameters. However, no significant improvement in retinal thinning or diabetic retinopathy-associated visual loss was found in treated diabetic retinas. Conclusions: Deep retinal microvasculature of diabetic Ins2Akita eyes shows late stage changes consistent with disorganized vascular proliferation. We show that intravitreally injected AAV2.COMP-Ang1 blocks this increase in deep microvascularity, even when administered subsequent to development of the first detectable vascular defects. However, improving vascular normalization did not attenuate neuroretinal degeneration or loss of visual acuity. Therefore, additional interventions are required to address neurodegenerative changes that are already underway.

Asunto(s)

Angiopoyetina 1/administración & dosificación , Proteína de la Matriz Oligomérica del Cartílago/administración & dosificación , Retinopatía Diabética/prevención & control , Vectores Genéticos , Parvovirinae/genética , Neovascularización Retiniana/prevención & control , Vasos Retinianos/efectos de los fármacos , Animales , Glucemia/metabolismo , Western Blotting , Capilares/efectos de los fármacos , Dependovirus , Diabetes Mellitus Tipo 1/complicaciones , Retinopatía Diabética/fisiopatología , Portadores de Fármacos , Combinación de Medicamentos , Femenino , Terapia Genética , Insulina/genética , Inyecciones Intravítreas , Masculino , Ratones , Ratones Endogámicos C57BL , Retina/patología , Neovascularización Retiniana/fisiopatología , Vasos Retinianos/patología , Agudeza Visual/fisiología

RESUMEN

Optical coherence tomography (OCT) angiography is a dye-free and non-invasive angiography which allows visualization of retinal and choroid vascular flow, enabling observation of highly permeable and three dimensional vasculature. Although OCT angiography is providing new insights in human retinal and choroidal diseases, a few studies have been reported in experimental mice. In this study, to determine the potential of OCT angiography in experimental mice, we sought to examine whether OCT angiography can detect vascular change in type I diabetic mice. To conduct age dependent analysis, 2 and 6 month old male type 1 diabetic Ins2Akita/+ and age matched C57BL/6J mice were used. OCT angiography was performed by Heidelberg Spectralis OCT Angiography Module with 30° lens + mouse adapter lens. We acquired the OCT angiography image from the peripheral nasal position. For analysis of OCT angiography images, OCT angiography positive area were used for vascular density. We analyzed vascular density from the retinal surface (inner limiting membrane) to 120 µm depth with 4 µm steps in order to correlate vascular density vs depth (N = 4 per group). Vascular density of both mouse strains demonstrated three different peaks. By comparing with the OCT image, the first peak (superficial), second peak (intermediate) and third peak (deep) were located in nerve fiber layer/ganglion cell layer, inner plexiform layer/inner nuclear layer and outer plexiform layer/outer nuclear layer, respectively. We calculated vascular density of these peaks separately. In C57BL/6J mice, the vascular density in all three layers do not show significant difference between 2- and 6-month-old. On the other hand, 6-month-old Ins2Akita/+ mice showed a significant decrease of the vascular density in all three layers compared to 2-month-old Ins2Akita/+ mice. Also, the vascular density of 6-month-old Ins2Akita/+ mice in the deep layer showed a significant decrease compared to 2- and 6-month-old C57BL/6J mice. Thus, OCT angiography successfully detects retinal vascular difference between type I diabetic mice and control mice, and age-dependent vasculature change in type I diabetic mice. The diabetic mice demonstrated reduced vascular density due to reduced density of flowing deep vessels. Importantly, we observed this difference without retinal blood leakage, hemorrhage or neovascularization. Our analysis (vascular density vs retinal depth) suggests that OCT angiography is useful for in vivo detection of retinal vasculature alteration in experimental mice.

Asunto(s)

Diabetes Mellitus Tipo 1/diagnóstico , Retinopatía Diabética/diagnóstico , Vasos Retinianos/patología , Envejecimiento/fisiología , Animales , Diabetes Mellitus Experimental/diagnóstico , Angiografía con Fluoresceína , Masculino , Ratones , Ratones Endogámicos C57BL , Microvasos/patología , Vasos Retinianos/diagnóstico por imagen , Tomografía de Coherencia Óptica

RESUMEN

Short-activating RNA (saRNA), which targets gene promoters, has been shown to increase the target gene expression. In this study, we describe the use of an saRNA (Flt a-1) to target the flt-1 promoter, leading to upregulation of the soluble isoform of Flt-1 and inhibition of angiogenesis. We demonstrate that Flt a-1 increased sFlt-1 mRNA and protein levels, while reducing VEGF expression. This was associated with suppression of human umbilical vascular endothelial cell (HUVEC) proliferation and cell cycle arrest at the G0/G1 phase. HUVEC migration and tube formation were also suppressed by Flt a-1. An siRNA targeting Flt-1 blocked the effects of Flt a-1. Flt a-1 effects were not mediated via argonaute proteins. However, trichostatin A and 5'-deoxy-5'-(methylthio) adenosine inhibited Flt a-1 effects, indicating that histone acetylation and methylation are mechanistically involved in RNA activation of Flt-1. In conclusion, RNA activation of sFlt-1 can be used to inhibit angiogenesis.

Asunto(s)

Proliferación Celular/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , ARN Bicatenario/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ciclo Celular/fisiología , Línea Celular , Movimiento Celular/fisiología , Metilación de ADN , Histonas/metabolismo , Humanos , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Regulación hacia Arriba , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética

RESUMEN

PURPOSE: We previously showed that intravitreal injection of the sFLT morpholino-oligomer (FLT-MO) suppresses laser-induced choroidal neovascularization (CNV) in mice by decreasing the membrane bound form of Flt-1 while increasing the soluble form of Flt-1 via alternative splicing shift. In this study, we examined whether cyclic RGD peptide (cRGD) can promote morpholino-oligomer accumulation in CNV following tail vein injection, and whether systemic cRGD conjugated FLT-MO (cRGD-FLT-MO) suppresses CNV growth. METHODS: cRGD conjugated fluorescent morpholino-oligomer (cRGD-F-MO) was injected via tail vein into mice with previous retinal laser photocoagulation and examined for cRGD-F-MO accumulation in CNV. To examine whether cRGD-FLT-MO suppresses CNV growth, mice were tail-vein injected with cRGD-FLT-MO, cRGD conjugated standard morpholino-oligomer (cRGD-STD-MO), or Dulbecco's Phosphate-Buffered Saline (DPBS) 1 and 4 days postlaser photocoagulation. Seven days postlaser photocoagulation, eyes were harvested and laser CNV was stained with isolectin GS-IB4, allowing quantification of CNV size by confocal microscopy. RESULTS: cRGD-F-MO accumulation in CNV commenced immediately after tail vein injection and could be observed even 1 day after injection. cRGD-FLT-MO tail vein injection significantly suppressed CNV size (2.7 × 105 ± 0.3 × 105 µm3, P < 0.05 by Student's t-test) compared with controls (DPBS: 5.1 × 105 ± 0.6 × 105 µm3 and cRGD-STD-MO: 5.5 × 105 ± 0.8 × 105 µm3). CONCLUSIONS: cRGD peptide facilitates morpholino-oligomer accumulation in CNV following systemic delivery. cRGD-FLT-MO suppressed CNV growth after tail-vein injection, demonstrating the potential utility of cRGD peptide for morpholino-oligomer delivery to CNV. TRANSLATIONAL RELEVANCE: Current therapy for neovascular age-related macular degeneration involves intravitreal injection of anti-vascular endothelial growth factor drugs. Our results indicate that CNV can be treated systemically, thus eliminating risks and hazards associated with intravitreal injection.

RESUMEN

Neovascular age-related macular degeneration (AMD) is treated with anti-VEGF intravitreal injections, which can cause geographic atrophy, infection, and retinal fibrosis. To minimize these toxicities, we developed a nanoparticle delivery system for recombinant Flt23k intraceptor plasmid (RGD.Flt23k.NP) to suppress VEGF intracellularly within choroidal neovascular (CNV) lesions in a laser-induced CNV mouse model through intravenous administration. In the current study, we examined the efficacy and safety of RGD.Flt23k.NP in mice. The effect of various doses was determined using fluorescein angiography and optical coherence tomography to evaluate CNV leakage and volume. Efficacy was determined by the rate of inhibition of CNV volume at 2 weeks post-treatment. RGD.Flt23k.NP had peak efficacy at a dose range of 30-60 µg pFlt23k/mouse. Using the lower dose (30 µg pFlt23k/mouse), RGD.Flt23k.NP safety was determined both in single-dose groups and in repeat-dose (three times) groups by measuring body weight, organ weight, hemoglobin levels, complement C3 levels, and histological changes in vital organs. Neither toxicity nor inflammation from RGD.Flt23k.NP was detected. No side effect was detected on visual function. Thus, systemic RGD.Flt23k.NP may be an alternative to standard intravitreal anti-VEGF therapy for the treatment of neovascular AMD.

Asunto(s)

Inhibidores de la Angiogénesis/administración & dosificación , Neovascularización Coroidal/terapia , Portadores de Fármacos , Degeneración Macular/terapia , Plásmidos/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Inhibidores de la Angiogénesis/química , Animales , Coroides/irrigación sanguínea , Coroides/metabolismo , Coroides/patología , Neovascularización Coroidal/genética , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Complemento C3/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Regulación de la Expresión Génica , Hemoglobinas/metabolismo , Humanos , Inyecciones Intravenosas , Inyecciones Intravítreas , Rayos Láser , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Nanopartículas/química , Plásmidos/química , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

PURPOSE: Clarithromycin is a 14-membered ring macrolide antibiotic with anti-inflammatory as well as antibacterial activity, and has been used worldwide. Moxifloxacin is a leading fourth generation quinolone antibiotic that has been used worldwide perioperatively. We intended to evaluate whether clarithromycin can suppress angiogenesis and inflammation in the cornea, and to compare the anti-inflammatory and anti-angiogenic effects of the two antibiotics, clarithromycin and moxifloxacin. METHODS: We made a murine corneal suture model and tested the anti-inflammatory and anti-angiogenic effects of clarithromycin (5 mg/ml) and moxifloxacin (5 mg/ml) in two randomly divided groups. Dexamethasone (5 mg/ml) was used as a positive control. After making two sutures on the cornea, we performed subconjunctival injections (10 µl) on each group on the day of suture, and every day thereafter until the 8th day post-suture. After harvesting corneas on the 8th post-suture day for immunohistochemical staining, we compared neovascularization (NV), lymphangiogenesis (LY) and inflammatory cell infiltration among the groups. RESULTS: Clarithromycin suppressed NV, LY and inflammatory infiltration, compared with phosphate-buffered saline (PBS). However, moxifloxacin did not suppress NV, LY, or inflammatory infiltration, compared with PBS. Comparison between clarithromycin and moxifloxacin, clarithromycin showed a tendency of decreasing LY (p = 0.063) and had less inflammatory cell infiltration (p < 0.05) than did the moxifloxacin group. The anti-(lymph)angiogenic and anti-inflammatory effects of clarithromycin were as high as those of dexamethasone. CONCLUSION: Clarithromycin suppressed LY and inflammation in the cornea, and its anti-inflammatory effect was significantly superior to that of moxifloxacin.

Asunto(s)

Claritromicina/administración & dosificación , Córnea/patología , Neovascularización de la Córnea/tratamiento farmacológico , Fluoroquinolonas/administración & dosificación , Queratitis/tratamiento farmacológico , Animales , Antibacterianos/administración & dosificación , Córnea/efectos de los fármacos , Neovascularización de la Córnea/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Inmunohistoquímica , Queratitis/patología , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Moxifloxacino

RESUMEN

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the U.S. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here, we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable with nondiabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR.

Asunto(s)

Angiopoyetina 1/uso terapéutico , Proteína de la Matriz Oligomérica del Cartílago/uso terapéutico , Diabetes Mellitus Tipo 1/complicaciones , Retinopatía Diabética/terapia , Modelos Animales de Enfermedad , Terapia Genética , Retina/patología , Angiopoyetina 1/química , Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Animales , Proteína de la Matriz Oligomérica del Cartílago/química , Proteína de la Matriz Oligomérica del Cartílago/genética , Proteína de la Matriz Oligomérica del Cartílago/metabolismo , Células Cultivadas , Terapia Combinada/efectos adversos , Cruzamientos Genéticos , Retinopatía Diabética/inmunología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Células Progenitoras Endoteliales/citología , Células Progenitoras Endoteliales/trasplante , Terapia Genética/efectos adversos , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Inyecciones Intravítreas , Leucocitos/citología , Leucocitos/inmunología , Leucocitos/metabolismo , Leucocitos/patología , Ratones Endogámicos C57BL , Ratones Mutantes , Estabilidad Proteica , Distribución Aleatoria , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Retina/inmunología , Retina/metabolismo , Solubilidad

RESUMEN

Long-term inhibition of extracellular vascular endothelial growth factor (VEGF) in the treatment of age-related macular degeneration (AMD) may induce retinal neuronal toxicity and risk other side effects. We developed a novel strategy which inhibits retinal pigment epithelium (RPE)-derived VEGF, sparing other highly sensitive retinal tissues. Flt23k, an intraceptor inhibitor of VEGF, was able to inhibit VEGF in vitro. Adeno-associated virus type 2 (AAV2)-mediated expression of Flt23k was maintained for up to 6 months postsubretinal injection in mice. Flt23k was able to effectively inhibit laser-induced murine choroidal neovascularization (CNV). VEGF levels in the RPE/choroid complex decreased significantly in AAV2.Flt23k treated eyes. Neither retinal structure detected by Heidelberg Spectralis nor function measured by electroretinography (ERG) was adversely affected by treatment with AAV2.Flt23k. Hence AAV2.Flt23k can effectively maintain long-term expression and inhibit laser-induced CNV in mice through downregulation of VEGF while maintaining a sound retinal safety profile. These findings suggest a promising novel approach for the treatment of CNV.

Asunto(s)

Neovascularización Coroidal/genética , Dependovirus/genética , Vectores Genéticos/genética , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas Recombinantes de Fusión , Transducción Genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Animales , Apoptosis , Coroides/metabolismo , Neovascularización Coroidal/patología , Neovascularización Coroidal/terapia , Modelos Animales de Enfermedad , Expresión Génica , Genes Reporteros , Terapia Genética , Vectores Genéticos/administración & dosificación , Humanos , Ratones , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/química

RESUMEN

PURPOSE: Wound induced corneal fibrosis can lead to permanent visual impairment. Keratocyte activation and differentiation play a key role in fibrosis, and vimentin, a major structural type III intermediate filament, is a required component of this process. The purpose of our study was to develop a nonviral therapeutic strategy for treating corneal fibrosis in which we targeted the knockdown of vimentin. METHODS: To determine the duration of plasmid expression in corneal keratocytes, we injected a naked plasmid expressing green fluorescent protein (GFP; pCMV-GFP) into an unwounded mouse corneal stroma. We then injected pCMV-GFP or plasmids expressing small hairpin RNA in the corneal wound injury model (full-thickness corneal incision) to evaluate opacification. RESULTS: GFP expression peaked between days 1 and 3 and had prominent expression for 15 days. In the corneal wound injury model, we found that the GFP-positive cells demonstrated extensive dendritic-like processes that extended to adjacent cells, whereas the vimentin knockdown model showed significantly reduced corneal opacity. CONCLUSIONS: These findings suggest that a nonviral gene therapeutic approach has potential for treating corneal fibrosis and ultimately reducing scarring.

Asunto(s)

Opacidad de la Córnea/genética , Opacidad de la Córnea/terapia , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , ARN/genética , Vimentina/uso terapéutico , Animales , Western Blotting , Células Cultivadas , Córnea/efectos de los fármacos , Córnea/metabolismo , Córnea/patología , Queratocitos de la Córnea/efectos de los fármacos , Queratocitos de la Córnea/metabolismo , Queratocitos de la Córnea/patología , Opacidad de la Córnea/patología , Modelos Animales de Enfermedad , Lesiones Oculares/metabolismo , Lesiones Oculares/patología , Femenino , Fibrosis/genética , Fibrosis/patología , Fibrosis/terapia , Estudios de Seguimiento , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la Polimerasa , Vimentina/biosíntesis , Vimentina/genética , Cicatrización de Heridas

RESUMEN

PURPOSE: Dry eye disease is becoming recognized as an immune-inflammation mediated disorder. Surgical insults such as corneal incision or suture can aggravate dry eye. We sought to determine whether underlying dry eye aggravates corneal inflammatory infiltration, hemangiogenesis, and lymphangiogenesis (LY) induced by surgical injury in a murine model. METHODS: We used treatment arms; one, normal eye (non-dry eye) and the other, a scopolamine-induced dry eye model. We first compared the corneas of both groups on which no surgery was performed with confocal and fluorescent microscopy. In subgroups of each treatment arm, we made a corneal incision followed by 2 corneal sutures to approximate the wound. After harvesting the cornea on postoperative day 9 and immunohistochemical staining, we compared corneal neovascularization (NV), LY, and CD11b inflammatory cell infiltration between non-dry eye and dry eye groups. RESULTS: In corneas in which no surgery was performed, the dry eye group showed more CD11b cell infiltration than did the non-dry eye group (P < 0.05). With respect to corneas after injury, there was significantly more hemangiogenesis, LY, and inflammatory infiltration in the dry eye group than in the non-dry eye group (all P < 0.05). CONCLUSIONS: The underlying status of the cornea, whether it is dry or not, plays a significant role in the development of NV, LY, and inflammation after corneal injury. Dry eye can aggravate post-injury NV, LY, and inflammation.

Asunto(s)

Lesiones de la Cornea , Neovascularización de la Córnea/etiología , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/complicaciones , Lesiones Oculares/complicaciones , Linfangiogénesis , Animales , Antígeno CD11b/metabolismo , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Síndromes de Ojo Seco/metabolismo , Síndromes de Ojo Seco/patología , Células Endoteliales/metabolismo , Femenino , Glicoproteínas/metabolismo , Queratitis/etiología , Queratitis/metabolismo , Queratitis/patología , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Microscopía Fluorescente , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Factores de Riesgo

RESUMEN

Monthly intraocular injections are widely used to deliver protein-based drugs that cannot cross the blood-retina barrier for the treatment of leading blinding diseases such as age-related macular degeneration (AMD). This invasive treatment carries significant risks, including bleeding, pain, infection, and retinal detachment. Further, current therapies are associated with a rate of retinal fibrosis and geographic atrophy significantly higher than that which occurs in the described natural history of AMD. A novel therapeutic strategy which improves outcomes in a less invasive manner, reduces risk, and provides long-term inhibition of angiogenesis and fibrosis is a felt medical need. Here we show that a single intravenous injection of targeted, biodegradable nanoparticles delivering a recombinant Flt23k intraceptor plasmid homes to neovascular lesions in the retina and regresses CNV in primate and murine AMD models. Moreover, this treatment suppressed subretinal fibrosis, which is currently not addressed by clinical therapies. Murine vision, as tested by OptoMotry, significantly improved with nearly 40% restoration of visual loss induced by CNV. We found no evidence of ocular or systemic toxicity from nanoparticle treatment. These findings offer a nanoparticle-based platform for targeted, vitreous-sparing, extended-release, nonviral gene therapy.

Asunto(s)

ADN/administración & dosificación , Terapia Genética/métodos , Degeneración Macular/terapia , Nanocápsulas/administración & dosificación , Neovascularización Patológica/terapia , Retina/patología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Animales , ADN/genética , Fibrosis , Haplorrinos , Ratones , Resultado del Tratamiento

RESUMEN

Delivery of anti-inflammatory steroids concurrently to both anterior and posterior segments of the eye is a challenge. The anterior ocular structures limit topical delivery. Injection can be disproportionately and repeatedly invasive and selective for only one ocular hemisphere. We developed a novel implant that can compensate for the limited conveyance of topical medicine and reduce the repetitive invasiveness of injection from the capsular bag allowing dexamethasone (DXM) delivery to both the anterior and posterior chambers. To establish proof of concept, microparticles were prepared with PLGA [poly(d,l-lactide-co-glycolide), 50:50, MW. 7000-17000], hydroxypropyl methyl cellulose (HPMC), and DXM by oil-in-water emulsion/solvent evaporation technique. Zeatsizer Nano and SEM (scanning electron microscopy) results showed microspheres in the range of 8±1µm. The target load of DXM in the microparticles was ~20.0% with a % recovery of 99.9% (w/w). Dose related pharmacokinetics with near zero order kinetics was observed for up to 6 weeks in rabbits with intracapsular bag implants. DXM flow was bidirectional from the endocapsular space and significant concentrations were found in the anterior and posterior chambers after up to 6 weeks. Whereas, with topical drops the exposure was minimal in all the ocular tissues with greater systemic exposure. Intraocular pressure was normal in all of the study groups; slit lamp biomicroscopy examinations revealed that no cells or fibrin formation in the anterior and posterior chamber with implants but flare, cells and fibrin was present in the topical drops group. Histological examination revealed normal tissues and no signs of inflammation in all the groups. The implant did not migrate to the center of the eye or obstruct the visual axis. We believe these findings demonstrate the feasibility of drug delivery from the capsular bag to the anterior and posterior segments effectively compared to topical alternatives.

Asunto(s)

Antiinflamatorios/administración & dosificación , Dexametasona/administración & dosificación , Implantes de Medicamentos/administración & dosificación , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacocinética , Catarata/tratamiento farmacológico , Catarata/metabolismo , Dexametasona/química , Dexametasona/farmacocinética , Implantes de Medicamentos/química , Implantes de Medicamentos/farmacocinética , Ojo/anatomía & histología , Ojo/efectos de los fármacos , Ojo/metabolismo , Femenino , Microesferas , Tamaño de la Partícula , Conejos , Uveítis/tratamiento farmacológico , Uveítis/metabolismo

RESUMEN

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.

Asunto(s)

Linfangiogénesis/genética , Morfolinos/genética , Neovascularización Fisiológica/genética , Empalme del ARN , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Animales , Secuencia de Bases , Trasplante de Córnea , Cartilla de ADN , Exones , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Microscopía Electrónica de Rastreo

RESUMEN

PURPOSE: This study sought to determine whether a Vascular Endothelial Growth Factor Receptor 1 (VEGFR1)-specific morpholino (MO) could decrease neovascularization, thereby enhancing murine cornea transplant survival, and if this effect is synergistic with steroid therapy. METHODS: Graft survival, corneal neovascularization, and corneal lymphangiogenesis were compared among the VEGFR1_MO, STD MO and PBS groups following subconjunctival injection in mice that underwent normal risk penetrating keratoplasty (NR PK) and high-risk penetrating keratoplasty (HR PK). Graft survival, corneal neovascularization, and corneal lymphangiogenesis in groups treated with both VEGFR1_MO and steroid therapy were also analyzed in HR PK. RESULTS: In NR PK, the VEGFR1_MO decreased angiogenesis, lymphangiogenesis, and increased graft survival compared with the PBS group (P = 0.055, P = 0.003, P = 0.043, respectively). In HR PK, VEGFR1_MO decreased angiogenesis, lymphangiogenesis, and increased graft survival compared with the STD MO (P = 0.000, P = 0.000, P = 0.029, respectively) and PBS groups (P = 0.004, P = 0.002, P = 0.024). In HR PK, when the VEGFR1_MO was combined with steroid therapy, a significant increase in graft survival was seen compared with steroid treatment alone (P = 0.045). The 2-month graft survival rate for HR PK was 27% in the combination group compared with 0% in the triamcinolone only group. CONCLUSIONS: VEGFR1_MO decreased angiogenesis and lymphangiogenesis, resulting in increased graft survival in both NR PK and HR PK. This beneficial effect is synergistically enhanced with steroid treatment in HR PK.

Asunto(s)

Neovascularización de la Córnea/prevención & control , Modelos Animales de Enfermedad , Supervivencia de Injerto/efectos de los fármacos , Queratoplastia Penetrante , Morfolinos/uso terapéutico , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Animales , Córnea/fisiología , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Sinergismo Farmacológico , Femenino , Glucocorticoides/uso terapéutico , Linfangiogénesis/efectos de los fármacos , Vasos Linfáticos/efectos de los fármacos , Vasos Linfáticos/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Triamcinolona Acetonida/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

PURPOSE: To evaluate the effect of glucocorticoid (triamcinolone acetonide injectable suspension) pretreatment on corneal neovascularization, lymphangiogenesis, and inflammation in a murine penetrating keratoplasty (PK) and corneal suture model. METHODS: For the PK model, BALB/c mice were used as recipients and C57BL/6 mice were used as donors. A group pretreated with subconjunctival glucocorticoid and a combination of post-subconjunctival and topical glucocorticoids (group I) was compared with two groups that did not receive glucocorticoid pretreatment [one group received a combination of subconjunctival and topical glucocorticoids postoperatively (group II) and the other group received only topical glucocorticoid treatment postoperatively (group III)]. All groups were treated with subconjunctival glucocorticoid on the day of surgery. For the corneal suture model, BALB/c mice were used. A group receiving only pre-suture glucocorticoid treatment (group A) and a group receiving only post-suture glucocorticoid treatment (group C) were compared with a control group that did not receive glucocorticoid therapy (group B). The degree of neovascularization, lymphangiogenesis, and inflammatory infiltration was compared in each of these models. RESULTS: In the PK model, the group receiving glucocorticoid pretreatment (group I) showed less neovascularization compared with the posttreatment-only groups (group II, P=0.043; group III, P=0.020) and less lymphangiogenesis compared with group III (P=0.005). In the corneal suture model, the glucocorticoid pretreatment group showed a similar level of neovascularization, lymphangiogenesis, and inflammatory infiltration as the posttreatment-only groups (P>0.05). CONCLUSIONS: Glucocorticoid pretreatment before PK decreases neovascularization and lymphangiogenesis compared with posttransplant glucocorticoid treatment alone.

Asunto(s)

Neovascularización de la Córnea/tratamiento farmacológico , Glucocorticoides/uso terapéutico , Queratoplastia Penetrante , Linfangiogénesis/efectos de los fármacos , Premedicación , Técnicas de Sutura , Triamcinolona Acetonida/uso terapéutico , Animales , Modelos Animales de Enfermedad , Supervivencia de Injerto/efectos de los fármacos , Presión Intraocular , Queratitis/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL

RESUMEN

PURPOSE: To determine if nanoparticles delivering plasmids expressing Flt23k (an anti-VEGF intraceptor) can enhance murine cornea transplant survival and whether their effect is synergistic with steroid therapy. METHODS: Biodegradable PLGA Flt23k loaded or blank nanoparticles were prepared using the emulsion solvent evaporation METHOD: Graft survival, corneal neovascularization, and corneal lymphangiogenesis were compared among the Flt23k nanoparticles, blank nanoparticles, triamcinolone acetonide, and PBS groups following subconjunctival injection in mice that underwent penetrating keratoplasty. Graft survival, corneal neovascularization, and corneal lymphangiogenesis in a group treated with both nanoparticles and steroid therapy were also analyzed. RESULTS: The Flt23k nanoparticle group showed less neovascularization, lymphangiogenesis, and graft failure compared with the PBS control group (P < 0.01). The 2-month graft survival rate was 20% in the Flt23k nanoparticle group with no grafts surviving in the PBS group. When the Flt23k nanoparticle was combined with steroid therapy, a significant increase in graft survival was seen compared with both steroid treatment alone (P < 0.05) and steroid combined with blank nanoparticle treatment (P < 0.05). The 2-month graft survival rate was 91.6% in the combination group compared with 47.6% in the triamcinolone-only group and 42.4% in the triamcinolone plus blank nanoparticle group. CONCLUSIONS: Anti-VEGF nanoparticles (Flt23k) have a significant effect on decreasing neovascularization and lymphangiogenesis, resulting in increased graft survival in penetrating keratoplasty. This beneficial effect is synergistically enhanced with steroid treatment.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Neovascularización de la Córnea/tratamiento farmacológico , Glucocorticoides/farmacología , Supervivencia de Injerto/efectos de los fármacos , Linfangiogénesis/efectos de los fármacos , Triamcinolona Acetonida/farmacología , Inhibidores de la Angiogénesis/genética , Animales , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Fluorescente , Nanopartículas , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores

RESUMEN

Current glaucoma management modalities are hindered by low patient compliance and adherence. This can be due to highly complex treatment strategies or poor patient understanding. Treatments focus on the management or reduction of intraocular pressure. This is most commonly done through the use of daily topical eye drops. Unfortunately, despite effective therapies, glaucoma continues to progress, possibly due to patients not adhering to their treatments. In order to mitigate these patient compliance issues, many sustained release treatments are being researched and are entering the clinic. Conjunctival, subconjunctival, and intravitreal inserts, punctal plugs, and drug depots are currently in clinical development. Each delivery system has hurdles, yet shows promise and could potentially mitigate the current problems associated with poor patient compliance.

RESUMEN

PURPOSE: This study sought to determine whether a vascular endothelial growth factor receptor 1 (VEGFR1)-specific morpholino could induce the alternative splicing of Flt-1 pre-mRNA to downregulate membrane-bound Flt-1 (mFlt-1) and increase the production of soluble Flt-1 (sFlt-1), thereby limiting angiogenesis and inflammation in a mouse corneal suture injury model. METHODS: A murine corneal suture model was used to investigate the effects of a VEGFR1-specific morpholino in vivo. Western blot analysis and RT-PCR were used to compare the impact of the Flt morpholino on mFlt-1 and sFlt-1 levels. For vascular regression modeling, two corneal sutures were placed and injected with Flt morpholino, standard morpholino, and PBS on days 8 and 10. Corneas were harvested on day 14. The grade of neovascularization (graded 0-5; 0, no neovascularization; 5, thick tortuous new vessel growth over the suture and toward the center of the cornea) was compared on days 8, 10, and 14. Immunohistochemistry, fluorescent microscopy, and confocal microscopy were used to digitally quantify the area and volume of neovascularization and inflammatory infiltration. RESULTS: Western blot analysis revealed that the Flt morpholino decreased mFlt-1 levels while increasing sFlt-1 levels. An increased sFlt-1/mFlt-1 ratio in the Flt morpholino group was seen with RT-PCR. Based on the neovascularization grading, there was a decrease in neovascularization area in the Flt morpholino group (3.29 ± 0.19 to 2.92 ± 0.13) from day 8 to 14 (P < 0.05) compared with that in both the standard morpholino (2.68 ± 0.19 to 3.14 ± 0.22) and in the PBS (2.96 ± 0.14 to 3.42 ± 0.19) groups, both of which showed an increase in neovascularization (P < 0.05). The Flt morpholino group also showed reduced neovascularization volume compared with that of the PBS (P = 0.001) and STD morpholino groups (P = 0.000). CONCLUSIONS: Flt morpholinos decrease mFlt-1 and increase sFlt-1 levels, resulting in decreased neovascularization in a murine corneal suture model.

Asunto(s)

Neovascularización de la Córnea/metabolismo , Morfolinos/farmacología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Western Blotting , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos BALB C , Precursores del ARN/genética , Precursores del ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Suturas , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética