RESUMEN
PURPOSE: To compare 2.0 mg ranibizumab (RBZ) injections with 0.5 mg RBZ for eyes with center-involved diabetic macular edema (DME) and a central subfield thickness (CFT) of ≥250 µm on time-domain optical coherence tomography.DesignRandomized, controlled, multicenter clinical trial. METHODS: Eligible eyes were randomized in a 1:1 ratio to 0.5 mg (n=77) or 2.0 mg (n=75) RBZ. Study eyes received 6-monthly injections.Main outcome measuresThe primary outcome measure was the mean change in best corrected visual acuity (BCVA) at month 6. Secondary outcomes included the incidence and severity of systemic and ocular adverse events and the mean change in CFT from baseline. RESULTS: In all, 152 eyes (152 patients) were randomized in the study. At month 6, the mean improvement from baseline BCVA was +9.43 letters in the 0.5 mg RBZ group and +7.01 letters in the 2.0 mg RBZ group (P=0.161). At month 6, one death occurred in the 0.5 mg RBZ group and three deaths in the 2.0 mg RBZ group, all due to myocardial infarction in subjects with a prior history of heart disease. Mean CFT was reduced by 168.58 µm in the 0.5 mg RBZ group and by 159.70 µm in the 2.0 mg RBZ group (P=0.708). CONCLUSIONS: There was no statistically significant difference in the mean number of letters gained between the 0.5 and 2.0 mg RBZ groups through month 6. In this DME study population, high-dose RBZ does not appear to provide additional benefit over 0.5 mg RBZ.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Retinopatía Diabética/tratamiento farmacológico , Edema Macular/tratamiento farmacológico , Ranibizumab/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Retinopatía Diabética/diagnóstico , Retinopatía Diabética/fisiopatología , Método Doble Ciego , Femenino , Estudios de Seguimiento , Humanos , Inyecciones Intravítreas , Edema Macular/diagnóstico , Edema Macular/fisiopatología , Masculino , Persona de Mediana Edad , Retina/patología , Tomografía de Coherencia Óptica , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
PURPOSE: To identify factors associated with visual outcomes in patients with diabetic macular edema (DME) treated with ranibizumab (RBZ) in the Ranibizumab for Edema of the mAcula in Diabetes-Protocol 2 (READ-2) Study. PATIENTS AND METHODS: Optical coherence tomography scans, fundus photographs, and fluorescein angiograms (FAs) were graded and along with baseline characteristics were correlated with month (M) 24 visual outcome of best-corrected visual acuity (BCVA) ≤20/100 (poor outcome) vs >20/100 (better outcome). RESULTS: Of 101 patients with a M20 visit or beyond, 27 (27%) had BCVA ≤20/100. Comparison of patients with or without poor outcome showed mean baseline BCVA of 16.8 letters (20/125) in the former compared with 30.4 letters (20/63; P<0.001). Mean change in BCVA between baseline and M24 was -2.6 letters in the poor outcome group compared with +9.8 letters (P<0.001). Foveal thickness (FTH) at M24 was 374.1 µm in the poor outcome group compared with 268.8 µm (P<0.01), a difference driven by 14 patients with mean FTH of 450.3 µm. Foveal atrophy occurred in 65% (11/17) in the poor outcome group compared with 17%(12/71, P=0.001). Persistent edema was noted in 52% (14/27) of patients with poor outcome. Laser scars near foveal center were significantly more common in patients with poor outcome who did not have edema vs those who did (78% (7/9) vs 23% (3/13) P=0.03). CONCLUSION: Poor baseline BCVA (≤20/125) in DME patients predicts poor visual outcome (≤20/100) after 2 years of treatment with RBZ and/or focal/grid laser, often due to foveal atrophy and/or persistent edema.
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Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Retinopatía Diabética/tratamiento farmacológico , Edema Macular/tratamiento farmacológico , Agudeza Visual/fisiología , Retinopatía Diabética/fisiopatología , Femenino , Angiografía con Fluoresceína , Humanos , Inyecciones Intravítreas , Edema Macular/fisiopatología , Masculino , Persona de Mediana Edad , Ranibizumab , Factores de Riesgo , Tomografía de Coherencia Óptica , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Diseases complicated by abnormal growth of vessels or excessive leakage are the most prevalent cause of moderate or severe vision loss in developed countries. Recent progress unraveling the molecular pathogenesis of several of these disease processes has led to new drug therapies that have provided major benefits to patients. However, those treatments often require frequent intraocular injections, and despite monthly injections, some patients have a suboptimal response. Gene transfer of antiangiogenic proteins is an alternative approach that has the potential to provide long-term suppression of neovascularization (NV) and/or excessive vascular leakage in the eye. Studies in animal models of ocular NV have demonstrated impressive results with a number of transgenes, and a clinical trial in patients with advanced neovascular age-related macular degeneration has provided proof-of-concept. Two ongoing clinical trials, one using an adeno-associated viral (AAV) vector to express a vascular endothelial growth factor-binding protein and another using a lentiviral vector to express endostatin and angiostatin, will provide valuable information that should help to inform future trials and provide a foundation on which to build.
Asunto(s)
Inhibidores de la Angiogénesis/genética , Ojo/irrigación sanguínea , Técnicas de Transferencia de Gen , Edema Macular/genética , Neovascularización Patológica/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Inhibidores de la Angiogénesis/uso terapéutico , Angiostatinas/genética , Ensayos Clínicos como Asunto , Dependovirus , Endostatinas/genética , Terapia Genética , Vectores Genéticos , Humanos , Edema Macular/terapia , Neovascularización Patológica/terapiaRESUMEN
AIM: The aim of the study was to assess the safety and bioactivity of a single intravitreal injection of vascular endothelial growth factor (VEGF) Trap-Eye in subjects with diabetic macular oedema (DMO). METHODS: Five subjects with DMO, foveal thickness > or =250 microm measured by optical coherence tomography (OCT), and best-corrected visual acuity (BCVA) between 20/40 and 20/320, were enrolled. Each participant received a single intravitreal injection of 4.0 mg of VEGF Trap-Eye followed by a 6-week observation period. Outcome measures included safety and biological activity, including changes in BCVA and excess retinal thickness assessed by OCT. RESULTS: Injections of VEGF Trap-Eye were well tolerated with no ocular toxicity. One patient had an unrelated serious adverse event: hospitalisation for cellulitis of the left foot 27 days after injection of VEGF Trap-Eye. Median baseline BCVA was 36 ETDRS letters read at 4 m (not ETDRS visual acuity score; Snellen equivalent: 20/50) and median baseline excess central 1 mm foveal thickness (FTH) was 108 microm. At 4 weeks after injection, the median excess FTH was 59 microm and the median improvement in BCVA was nine letters. At 6 weeks after injection, four of the five patients showed improvement in excess FTH (median 74 microm; 31% reduction from baseline, p = 0.0625) and four of the five showed improvement in BCVA (median improvement of three letters). CONCLUSIONS: A single intravitreal injection of 4.0 mg of VEGF Trap-Eye was well tolerated and preliminary evidence of bioactivity was detected. These findings support additional studies investigating multiple injections of VEGF Trap-Eye in patients with DMO.
Asunto(s)
Retinopatía Diabética/tratamiento farmacológico , Edema Macular/tratamiento farmacológico , Proteínas Recombinantes de Fusión/efectos adversos , Anciano , Retinopatía Diabética/patología , Retinopatía Diabética/fisiopatología , Femenino , Angiografía con Fluoresceína , Humanos , Inyecciones , Edema Macular/patología , Edema Macular/fisiopatología , Masculino , Persona de Mediana Edad , Proyectos Piloto , Estudios Prospectivos , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/uso terapéutico , Retina/patología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Agudeza Visual/efectos de los fármacos , Cuerpo VítreoRESUMEN
Epidemiologic studies have suggested that elderly patients who consumed diets rich in antioxidants throughout their lives are less likely to be afflicted with age-related macular degeneration (AMD). This led to the Age-Related Eye Disease Study, which showed that supplements containing antioxidant vitamins and zinc reduce the risk of progression to severe stages of AMD. Despite these data that indirectly implicate oxidative damage in the pathogenesis of AMD, there has not been any direct demonstration of increased oxidative damage in the retinas of patients with AMD. In this study, we used biomarkers of oxidative damage in postmortem eyes from patients with AMD and comparably aged patients without AMD to directly assess for oxidative damage. Sections from 4 eyes with no pathologic features of AMD showed no immunofluorescent staining for markers of oxidative damage, while sections from 8 of 12 eyes with advanced geographic atrophy showed evidence of widespread oxidative damage in both posterior and anterior retina. Only 2 of 8 eyes with choroidal neovascularization and 2 of 16 eyes with diffuse drusen and no other signs of AMD showed evidence of oxidative damage. These data suggest that widespread oxidative damage occurs in the retina of some patients with AMD and is more likely to be seen in patients with advanced geographic atrophy. This does not rule out oxidative damage as a pathogenic mechanism in patients with CNV, but suggests that a subpopulation of patients with geographic atrophy may have a major deficiency in the oxidative defense system that puts the majority of cells in the retina at risk for oxidative damage.
Asunto(s)
Envejecimiento , Antioxidantes/farmacología , Degeneración Macular/metabolismo , Degeneración Macular/patología , Acroleína/metabolismo , Anciano , Anciano de 80 o más Años , Neovascularización Coroidal , Femenino , Humanos , Inmunohistoquímica , Degeneración Macular/prevención & control , Masculino , Microscopía Fluorescente , Estrés Oxidativo , Retina/metabolismo , Retina/patología , Factores de RiesgoRESUMEN
The CMV promoter drives high transgene expression and is one of the most commonly used promoters for gene transfer. Tissue-specific mammalian promoters provide an alternative, and it would be useful to have a system to directly compare them to viral promoters free from potential confounding vector-related effects. In this study, we describe how electroporation after subretinal injection of plasmid DNA can be used to perform comparative quantitative analysis of promoter activities. Luciferase assay of eyecup homogenates was carried out after coinjection/electroporation of pGL2, a plasmid containing the promoter fragment of interest coupled to the firefly luciferase gene, and pRL-CMV, a plasmid containing the CMV promoter coupled to the Renilla luciferase gene for normalization. This technique was used to compare activity of different fragments of the 5'-upstream region of the vitelliform macular dystrophy 2 (VMD2) gene, which is selectively expressed in the retinal pigmented epithelial (RPE) cells, and results indicated positive regulatory elements between -104 and -154 bp and between -424 and -585 bp. Addition of a fragment from intron 1 reduced the activity of the -585/+38 bp fragment by 75%. Deletion analysis implicated a 342 bp region near the 5'-end of intron 1 in the repression. Results of transient transfections in two cell lines that constitutively express VMD2 were similar, and results in transgenic mice were consistent, providing validation for promoter analysis by in vivo electroporation. We then explored the time course of expression of the -585/+38 VMD2 promoter fragment and found that compared to cassettes driven by CMV or SV40 promoters, which showed peak luciferase activity on day 2 followed by a rapid decrease in activity, the VMD2 promoter fragment showed lower activity initially, but the activity was sustained for up to 56 days (longest time point measured). A promoter fragment from another RPE-specific gene, Rpe65, showed a similar pattern of sustained expression for at least 112 days. These data indicate that nonviral gene transfer can be used to quantitatively evaluate the activity of promoter fragments independent of influence from viral vectors. A potentially important finding using this new technique is the demonstration that relatively sustained passenger gene expression can be achieved with nonviral gene transfer using mammalian rather than viral promoters.
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Electroporación , Oftalmopatías/terapia , Proteínas del Ojo/genética , Terapia Genética/métodos , Epitelio Pigmentado Ocular/metabolismo , Regiones Promotoras Genéticas , Animales , Bestrofinas , Canales de Cloruro , Citomegalovirus/genética , Expresión Génica , Ingeniería Genética , Humanos , Luciferasas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Plásmidos , Factores de TiempoRESUMEN
The eye is a relatively isolated tissue compartment, which provides advantages for utilization of small interfering RNA (siRNA). Feasibility of using siRNA for treatment of choroidal neovascularization has been demonstrated using siRNA directed against vascular endothelial growth factor (VEGF) or VEGF receptor 1 (VEGFR1), and both of these approaches are being tested in clinical trials. The results with VEGFR1 siRNA show that VEGFR1 is pro-angiogenic in the eye and is not a decoy receptor as it is in developmental angiogenesis. Topical delivery of siRNAs directed against VEGF or its receptors has also been shown to suppress corneal neovascularization. Signaling through transforming growth factor-beta receptor 2 (TGFbetaR2) has been implicated in excessive ocular scarring and TGFbetaR2 siRNA has shown benefit in a model relevant to excessive scarring after glaucoma filtration surgery. RNAi has been used to identify genes that promote apoptosis or oxidative damage in retinal cells and could be the basis of new treatments for glaucoma or photoreceptor degenerations. In cultured cells derived from ocular tissues, siRNA has become a valuable tool to explore the potential role of various genes in ocular disease processes. Based upon this early experience in vivo and in vitro, it appears that siRNAs may be valuable to help define the pathogenesis and develop new treatments for several ocular diseases.
Asunto(s)
Oftalmopatías/etiología , Oftalmopatías/terapia , Terapia Genética/métodos , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Animales , Humanos , Proyectos de InvestigaciónRESUMEN
In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth factor receptor 1 (vegfr1) mRNA to investigate the role of VEGFR1 in ocular neovascularization (NV). After evaluating many siRNAs, Sirna-027 was identified; it cleaved vegfr1 mRNA at the predicted site and reduced its levels in cultured endothelial cells and in mouse models of retinal and choroidal neovascularization (CNV). Compared to injection of an inverted control sequence, quantitative reverse transcriptase-PCR demonstrated statistically significant reductions of 57 and 40% in vegfr1 mRNA after intravitreous or periocular injection of Sirna-027, respectively. Staining showed uptake of 5-bromodeoxyuridine-labeled Sirna-027 in retinal cells that lasted between 3 and 5 days after intravitreous injection and was still present 5 days after periocular injection. In a CNV model, intravitreous or periocular injections of Sirna-027 resulted in significant reductions in the area of NV ranging from 45 to 66%. In mice with ischemic retinopathy, intravitreous injection of 1.0 mug of Sirna-027 reduced retinal NV by 32% compared to fellow eyes treated with 1.0 mug of inverted control siRNA. These data suggest that VEGFR1 plays an important role in the development of retinal and CNV and that targeting vegfr1 mRNA with siRNA has therapeutic potential.
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Neovascularización Coroidal/terapia , Terapia Genética/métodos , ARN Mensajero/genética , ARN Interferente Pequeño/administración & dosificación , Neovascularización Retiniana/terapia , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Animales , Células Cultivadas , Neovascularización Coroidal/metabolismo , Células Endoteliales/metabolismo , Inyecciones , Ratones , Ratones Endogámicos , Modelos Animales , ARN Mensajero/análisis , Neovascularización Retiniana/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/fisiología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoAsunto(s)
Anticuerpos Monoclonales/uso terapéutico , Neovascularización Coroidal/tratamiento farmacológico , Miopía Degenerativa/complicaciones , Adulto , Anticuerpos Monoclonales Humanizados , Bevacizumab , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
In this study, we explored the use of electroporation or media that promote lipoplex formation for nonviral gene transfer in the eye. There was no detectable staining for LacZ after subretinal, intravitreous, or periocular injection of a plasmid containing a CMV promoter expression cassette for LacZ, but when plasmid injection in each of the three sites was combined with electroporation, there was efficient transduction. Specific staining for LacZ was seen in retinal pigmented epithelial (RPE) cells after subretinal injection of a plasmid containing a vitelliform macular dystrophy 2 (VMD2) promoter expression cassette, demonstrating that this approach can be used to evaluate purported tissue-specific promoters in vivo. Electroporation with 10 V/mm resulted in strong LacZ staining, but was damaging to photoreceptors; substantial transduction with no evidence of retinal damage was seen using 3.4 V/mm. Staining for LacZ was also seen after subretinal or periocular, but not intravitreous, injection of plasmid DNA in medium containing 40% Lipofectamine2000 (Lf). Injection of 40% Lf into the subretinal space caused damage to photoreceptors, but subretinal injection of plasmid DNA in medium containing 10% NeuroPorter resulted in transduction of RPE cells with no adverse effects on retinal morphology or function as assessed by electroretinograms (ERGs). After either electroporation or lipofection, LacZ staining was detectable for at least 14 days, and could be reinduced by a second procedure. These data suggest that electroporation or lipofection can be used as experimental tools for ocular gene transfer to evaluate tissue-specific promoter fragments or to evaluate the effects of transgene expression in the retina. Also, with additional optimization, nonviral gene transfer may prove to be a valuable approach for the treatment of retinal and choroidal diseases.
Asunto(s)
Electroporación/métodos , Oftalmopatías/terapia , Terapia Genética/métodos , Epitelio Pigmentado Ocular/metabolismo , Animales , Electrorretinografía , Expresión Génica , Histocitoquímica/métodos , Inyecciones , Operón Lac , Lípidos/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Músculos Oculomotores/metabolismo , Plásmidos/administración & dosificación , Células Ganglionares de la Retina/metabolismo , Factores de TiempoRESUMEN
Several retinal and choroidal diseases are potentially treatable by intraocular delivery of genes whose products may counter or neutralize abnormal gene expression that occurs as part of the diseases. However, prior to considering a transgene, it is necessary to thoroughly investigate the effects of its expression in normal and diseased eyes. An efficient way to do this is to combine tissue-specific promoters with inducible promoter systems in transgenic mice. In this study, we used this approach to evaluate the effects of ectopic expression of angiopoietin-1 (Ang1) in normal eyes and those with ocular neovascularization. Adult mice with induced expression of Ang1 ubiquitously, or specifically in the retina, appeared normal and had no identifiable changes in retinal or choroidal blood vessels or in retinal function as assessed by electroretinography. Increased expression of Ang1 in eyes with severe retinal ischemia or in eyes with rupture of Bruch's membrane significantly suppressed the development of retinal or choroidal neovascularization, respectively. This inhibition of ocular neovascularization is particularly interesting and noteworthy, because overexpression of Ang1 in skin stimulates neovascularization. Ang1 also significantly reduced VEGF-induced retinal vascular permeability. These data suggest that intraocular delivery of ang1 has potential for treatment of ocular neovascularization and macular edema.
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Angiopoyetina 1/fisiología , Barrera Hematorretinal/fisiología , Neovascularización Coroidal/fisiopatología , Neovascularización Retiniana/fisiopatología , Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Animales , Neovascularización Coroidal/etiología , Neovascularización Coroidal/metabolismo , Isquemia/complicaciones , Isquemia/metabolismo , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Retina/metabolismo , Neovascularización Retiniana/etiología , Neovascularización Retiniana/metabolismo , Vasos Retinianos/fisiopatologíaRESUMEN
Gene transfer provides an exciting new approach for the treatment of retinal and choroidal diseases. Two areas of concern are the potential for vector-related toxicity and uncertainties associated with prolonged transgene expression. One way to address these concerns for transfer of genes encoding secreted proteins is to transduce cells on the outside of the eye, provided the gene product can gain access to the eye and have the desired effect. In this study, we investigated the feasibility of this approach. Periocular injection of an adenoviral vector encoding beta-galactosidase (AdLacZ.10) resulted in LacZ-stained cells throughout the orbit and around the eye. Compared to periocular injection of 5 x 10(9) particles of control vector, periocular injection of 5 x 10(9) or 1 x 10(9) particles of an adenoviral vector expressing pigment epithelium-derived factor (PEDF) regulated by a CMV promoter (AdPEDF.11) resulted in significantly elevated intraocular levels of PEDF and suppression of choroidal neovascularization. Periocularly injected recombinant PEDF was also found to diffuse through the sclera into the eye. Although similar experiments are needed in an animal with a human-sized eye, these data suggest that periocular gene transfer deserves consideration for the treatment of choroidal diseases.
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Adenoviridae/genética , Neovascularización Coroidal/terapia , Proteínas del Ojo , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Factores de Crecimiento Nervioso , Proteínas/genética , Serpinas/genética , Animales , Ojo/metabolismo , Expresión Génica , Inyecciones , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Órbita/metabolismo , Proteínas/metabolismo , Serpinas/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Several types of insult cause up-regulation of neurotrophic factors and their receptors in the retina resulting in decreased photoreceptor cell death from subsequent injury. This phenomenon is more prominent in rats than in mice and neurotrophic factors are more efficacious in rats than mice. If up-regulation of neurotrophic factor receptors on photoreceptor cells early in the course of degenerations contributes to neurotrophic factor survival-promoting activity, it may also increase the ability to detect neurotrophic factor-induced signaling in photoreceptors, particularly in rats. In this study, these hypotheses were investigated by performing immunohistochemical staining for the phosphorylated form of extracellular receptor kinase (pERK) or c-fos after intravitreous injection of neurotrophic factors in wild type rats or mice, or those with inherited retinal degenerations. In both rats and mice either early or late in the course of degeneration, or in wild type animals, intravitreous injection of brain-derived neurotrophic factor, ciliary neurotrophic factor, or fibroblast growth factor-2 caused immunostaining for pERK and c-fos in cells of the inner retina, particularly Müller cells, but not in photoreceptors. These data add to the mounting evidence suggesting that neurotrophic factors act indirectly through Müller cells to promote photoreceptor survival.
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Comunicación Celular/fisiología , Factores de Crecimiento Nervioso/metabolismo , Degeneración Retiniana/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Ciliar/metabolismo , Modelos Animales de Enfermedad , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-DawleyRESUMEN
Fibroblast growth factor-2 (FGF2) has neurotrophic effects in vitro and in vivo. It has been demonstrated to decrease photoreceptor cell death in rats exposed to constant light and in rats with an inherited defect in retinal pigmented epithelium (RPE) phagocytosis, but the effects of intravitreous injections of FGF2 in mice are equivocal. In this study, we used transgenic mice with increased expression of FGF2 in photoreceptors (rhodopsin promoter/FGF2 transgenics) to investigate the effects of sustained increased expression of FGF2 in mice with various types of photoreceptor degeneration, including rd mice that are homozygous for mutated phosphodiesterase beta subunit, Q344ter mice that undergo photoreceptor degeneration because of expression of mutated rhodopsin, and mice exposed to 75% oxygen for 1 or 2 weeks. At P21, the outer nuclear layer was markedly reduced in rd mice or Q344ter mice regardless of whether they inherited the rhodopsin promoter/FGF2 transgene. However, after 2 weeks of exposure to 75% oxygen, outer nuclear layer thickness was significantly reduced in littermate control mice compared to FGF2 transgenic mice (P = 0.0001). These data indicate that increased expression of FGF2 in photoreceptors protects them from hyperoxia-induced damage, but does not decrease cell death related to expression of mutated proteins involved in the phototransduction pathway. This suggests that FGF2 protects photoreceptors from oxidative damage, which may play a role in complex genetic diseases such as age-related macular degeneration.
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Factor 2 de Crecimiento de Fibroblastos/farmacología , Hiperoxia/fisiopatología , Células Fotorreceptoras de Vertebrados/fisiología , Envejecimiento/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Muerte Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos/genética , Mutación , ARN Mensajero/metabolismo , Valores de Referencia , Degeneración Retiniana/genética , Degeneración Retiniana/fisiopatología , Rodopsina/genéticaRESUMEN
Endostatin is a cleavage product of collagen XVIII that inhibits tumor angiogenesis and growth. Interferon alpha2a blocks tumor angiogenesis and causes regression of hemangiomas, but has no effect on choroidal neovascularization (CNV). Therefore, inhibitors of tumor angiogenesis do not necessarily inhibit ocular neovascularization. In this study, we used an intravenous injection of adenoviral vectors containing a sig-mEndo transgene consisting of murine immunoglobulin kappa-chain leader sequence coupled to sequence coding for murine endostatin to investigate the effect of high serum levels of endostatin on CNV in mice. Mice injected with a construct in which sig-mEndo expression was driven by the Rous sarcoma virus promoter had moderately high serum levels of endostatin and significantly smaller CNV lesions at sites of laser-induced rupture of Bruch's membrane than mice injected with null vector. Mice injected with a construct in which sig-mEndo was driven by the simian cytomegalovirus promoter had approximately 10-fold higher endostatin serum levels and had nearly complete prevention of CNV. There was a strong inverse correlation between endostatin serum level and area of CNV. This study provides proof of principle that gene therapy to increase levels of endostatin can prevent the development of CNV and may provide a new treatment for the leading cause of severe loss of vision in patients with age-related macular degeneration.
Asunto(s)
Inhibidores de la Angiogénesis/genética , Coroides/irrigación sanguínea , Colágeno/genética , Terapia Genética/métodos , Neovascularización Patológica/prevención & control , Fragmentos de Péptidos/genética , Adenoviridae/genética , Inhibidores de la Angiogénesis/sangre , Animales , Colágeno/sangre , Colágeno Tipo XVIII , Endostatinas , Expresión Génica , Genes Reporteros/genética , Vectores Genéticos , Inyecciones Intravenosas , Hígado/fisiología , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/patología , Fenómenos Fisiológicos Oculares , Fragmentos de Péptidos/sangre , Recombinación GenéticaRESUMEN
In this study, we investigated whether overexpression of pigment epithelium-derived factor (PEDF) by gene transfer can inhibit neovascularization by testing its effect in three different models of ocular neovascularization. Intravitreous injection of an adenoviral vector encoding PEDF resulted in expression of PEDF mRNA in the eye measured by RT-PCR and increased immunohistochemical staining for PEDF protein throughout the retina. In mice with laser-induced rupture of Bruch's membrane, choroidal neovascularization was significantly reduced after intravitreous injection of PEDF vector compared to injection of null vector or no injection. Subretinal injection of the PEDF vector resulted in prominent staining for PEDF in retinal pigmented epithelial cells and strong inhibition of choroidal neovascularization. In two models of retinal neovascularization (transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors and mice with oxygen-induced ischemic retinopathy), intravitreous injection of null vector resulted in decreased neovascularization compared to no injection, but intravitreous injection of PEDF vector resulted in further inhibition of neovascularization that was statistically significant. These data suggest that sustained increased intraocular expression of PEDF by gene therapy might provide a promising approach for treatment of ocular neovascularization.
Asunto(s)
Coroides/irrigación sanguínea , Proteínas del Ojo , Neovascularización Fisiológica/fisiología , Factores de Crecimiento Nervioso , Proteínas/genética , Retina/fisiología , Serpinas/genética , Adenoviridae/genética , Animales , Anticuerpos , Humor Acuoso , Coroides/química , Coroides/fisiología , Factores de Crecimiento Endotelial/genética , Expresión Génica/fisiología , Técnicas de Transferencia de Gen , Humanos , Inmunohistoquímica , Linfocinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas/análisis , Proteínas/inmunología , ARN Mensajero/análisis , Conejos , Retina/química , Serpinas/análisis , Serpinas/inmunología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Bovine Kir7.1 clones were obtained from a retinal pigment epithelium (RPE)-subtracted cDNA library. Human RPE cDNA library screening resulted in clones encoding full-length human Kir7.1. Northern blot analysis indicated that bovine Kir7.1 is highly expressed in the RPE. Human Kir7.1 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. The macroscopic Kir7.1 conductance exhibited mild inward rectification and an inverse dependence on extracellular K+ concentration ([K+]o). The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.013) > Na+ (0.003) approximately Li+ (0.001) and the sequence based on conductance ratios was Rb+ (9.5) >> K+ (1.0) > Na+ (0.458) > Cs+ (0.331) > Li+ (0.139). Non-stationary noise analysis of Rb+ currents in cell-attached patches yielded a unitary conductance for Kir7.1 of approximately 2 pS. In whole-cell recordings from freshly isolated bovine RPE cells, the predominant current was a mild inwardly rectifying K+ current that exhibited an inverse dependence of conductance on [K+]o. The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.021) > Na+ (0.003) approximately Li+ (0.002) and the sequence based on conductance ratios was Rb+ (8.9) >> K+ (1.0) > Na+ (0.59) > Cs+ (0.23) > Li+ (0.08). In cell-attached recordings with Rb+ in the pipette, inwardly rectifying currents were observed in nine of 12 patches of RPE apical membrane but in only one of 13 basolateral membrane patches. Non-stationary noise analysis of Rb+ currents in cell-attached apical membrane patches yielded a unitary conductance for RPE Kir of approximately 2 pS. On the basis of this molecular and electrophysiological evidence, we conclude that Kir7.1 channel subunits comprise the K+ conductance of the RPE apical membrane.
Asunto(s)
Epitelio Pigmentado Ocular/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/metabolismo , Animales , Artefactos , Northern Blotting , Bovinos , Membrana Celular/metabolismo , Clonación Molecular , Conductividad Eléctrica , Humanos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Permeabilidad , Canales de Potasio/fisiología , Distribución Tisular , XenopusRESUMEN
Proteolysis of vascular basement membranes and surrounding extracellular matrix is a critical early step in neovascularization. It requires alteration of the balance between matrix metalloproteinases (MMPs) and proteins that bind to and inactivate MMPs, tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 has been demonstrated to inhibit neovascularization in chick chorioallantoic membranes. However, TIMP-1 has also been shown to either promote or inhibit cell proliferation and migration in different settings. To determine whether genetic alteration of the MMP/TIMP-1 ratio would alter retinal neovascularization, we crossed mice that express vascular endothelial growth factor (VEGF) in photoreceptors with TIMP-1-deficient mice or mice that overexpress TIMP-1. Compared to VEGF transgene-positive/TIMP-1-sufficient mice, VEGF transgene-positive/TIMP-1-deficient mice showed smaller neovascular lesions. There was also no difference between the two groups of mice in the appearance of the neovascularization by light or electron microscopy. Compound VEGF/TIMP-1 transgenic mice had increased expression of both VEGF and TIMP-1 in the retina, and had more neovascularization than mice that had increased expression of VEGF alone. These gain- and loss-of-function data suggest that alteration of the TIMP-1/MMP ratio modulates retinal neovascularization in a complex manner and not simply by altering the proteolytic activity and thereby invasiveness of endothelial cells.
Asunto(s)
Factores de Crecimiento Endotelial/farmacología , Linfocinas/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Vasos Retinianos/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/fisiología , Actinas/biosíntesis , Actinas/genética , Animales , Northern Blotting , Southern Blotting , Sinergismo Farmacológico , Ratones , Ratones Transgénicos , Microscopía Electrónica , Neovascularización Fisiológica/genética , Vasos Retinianos/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor Tisular de Metaloproteinasa-1/deficiencia , Inhibidor Tisular de Metaloproteinasa-1/genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
PURPOSE: To assess subjective levels of eye pain, nausea, and sedation following vitreoretinal surgery performed with intravenous sedation and retrobulbar anesthesia. METHODS: One hundred eighty-five consecutive patients who underwent vitreoretinal surgery and had access to a standard postoperative analgesic regimen prospectively quantified levels of eye pain, nausea, and sedation 2 hours and 5 hours after surgery by using a standard visual analog scale. Analgesic requests were recorded. Responses were statistically analyzed. RESULTS: Fifty-six percent of patients had some eye pain after vitreoretinal surgery; 48% of patients requested an analgesic within 5 hours after surgery. Twenty-seven percent of patients required narcotic analgesia. There was a significant relationship between the presence of eye pain and surgery duration of >2 hours (P < 0.02). Sixteen percent of patients had postoperative nausea, which more likely occurred in those who received a narcotic analgesic (P < 0.02). Eighty percent of patients had postoperative sedation, which more likely occurred in those who received a narcotic analgesic (P < 0.02). CONCLUSION: One half of individuals undergoing vitreoretinal surgery, especially those who have lengthy procedures (>2 hours), will request pain medication within 5 hours after surgery; one half of these patients will need narcotic analgesia for pain control. Narcotics may result in nausea and sedation.
Asunto(s)
Oftalmopatías/cirugía , Dolor Postoperatorio/diagnóstico , Enfermedades de la Retina/cirugía , Cuerpo Vítreo/cirugía , Anciano , Analgésicos/administración & dosificación , Anestesia Intravenosa/métodos , Anestesia Local/métodos , Femenino , Humanos , Hipnóticos y Sedantes/administración & dosificación , Masculino , Persona de Mediana Edad , Náusea/diagnóstico , Náusea/tratamiento farmacológico , Náusea/etiología , Dimensión del Dolor , Dolor Postoperatorio/tratamiento farmacológico , Dolor Postoperatorio/etiología , Estudios Prospectivos , Factores de TiempoRESUMEN
PURPOSE: Vascular endothelial growth factor (VEGF) is upregulated by hypoxia and is a major stimulatory factor for retinal neovascularization in ischemic retinopathies such as diabetic retinopathy. This study sought to determine if VEGF is a stimulatory factor in a murine model of choroidal neovascularization (CNV). METHODS: Mice with laser-induced ruptures in Bruch's membrane were treated with vehicle alone; a drug that inhibits both VEGF and platelet-derived growth factor (PDGF) receptor kinases; a drug that inhibits PDGF, but not VEGF receptor kinase; or genistein, a nonspecific kinase inhibitor. After two weeks, CNV was quantified and compared. RESULTS: Blockade of phosphorylation by VEGF and PDGF receptors caused dramatic, almost complete inhibition of CNV. Genistein also had an inhibitory effect, but less so than the VEGF/PDGF receptor blocker. Blockade of phosphorylation by PDGF receptors, but not VEGF receptors, had no significant effect on CNV. CONCLUSIONS: These data and our previous study, which demonstrated that a kinase inhibitor that blocks VEGF and PDGF receptors and several isoforms of protein kinase C causing dramatic inhibition of CNV, suggest that VEGF signaling plays a critical role in the development of CNV in this model. If safety is established, the effect of inhibiting VEGF receptor kinase activity should be investigated in patients with CNV.