RESUMEN
We examined differently aged postnatal (P) rats for the distribution and uptake of iron in the eye with the main emphasis on iron uptake in the retina. The concentration of iron in the eye was 48 µg/g in rats aged one postnatal day (P1). Then concentration fell to approximately 12 µg/g at P30 and rose to 35 µg/g at P70. Perls' stain which labels both ferrous and ferric iron was found to exhibit a weak labeling in the retina at P1 contrasted by a robust labeling of macrophages found in the choroid of the retina. In older aged rats, the labeling of cells of the retina was much more intense and confined to cells widespread in the layers of the retina. In both P16 and adult rats injected intravenously with [(59)Fe-(125)I]transferrin, the uptake of (59)Fe, estimated as the volume of distribution, was significantly higher than that of [(125)I]transferrin, and uptake of both compounds was higher than that of simultaneously injected [(131)I]albumin. In the P16 rat, the uptake of (59)Fe expressed as the volume of distribution, V(D), rose linearly reaching approximately 2500 nl at 60 min. In the adult rat, the uptake of (59)Fe was of the same magnitude. Comparing P1 and P16 rats, the uptake of (59)Fe, [(125)I]transferrin and [(131)I]albumin was higher at P1 in both eyeball and retina. Emulsion autoradiography of retinas from P16 and adult rats injected with [(55)Fe]transferrin into the vitreous body showed uptake mainly in photoreceptor cells and retinal ganglion cells. Adult rats injected into the vitreous body with [(59)Fe]transferrin showed anterograde axonal transport from the retina into the optic nerve, optic tract, and superior colliculus. Immunoprecipitates of homogenates of the optic nerve revealed that (59)Fe was precipitable with an antibody raised against ferritin, indicative of detachment of iron from transferrin within the axons of the retinal ganglion cells. The data demonstrate an age-dependent but continuous iron uptake by the retina, and are indicative of anterograde axonal transport of transferrin by retinal ganglion cells.
Asunto(s)
Transporte Axonal/fisiología , Hierro/metabolismo , Retina/citología , Retina/crecimiento & desarrollo , Retina/metabolismo , Albúminas/metabolismo , Animales , Autorradiografía , Ojo/anatomía & histología , Ojo/crecimiento & desarrollo , Hierro/química , Radioisótopos/metabolismo , Ratas , Ratas Wistar , Transferrina/química , Transferrina/metabolismoRESUMEN
VEGF is considered as an important factor in the pathogenesis of macular edema. VEGF induces the rupture of the blood retinal barrier and may also influence the retinal pigment epithelial (RPE) outer retinal barrier. The aim of this work was to analyze the influence of the VEGF receptor pathways in the modulation of the RPE barrier breakdown in vitro and in vivo. The ARPE19 human junctions in culture are modulated by VEGF through VEGFR-1 but not through VEGFR-2. PlGF-1, that is a pure agonist of VEGFR-1, is produced in ARPE-19 cells under hypoxic conditions and mimics VEGF effects on the external retinal barrier as measured by TER and inulin flux. In vivo, the intravitreous injection of PlGF-1 induces a rupture of the external retinal barrier together with a retinal edema. This effect is reversible within 4 days. VEGF-E, that is a pure agonist of VEGFR-2, does not induce any acute effect on the RPE barrier. These results demonstrate that PlGF-1 can reproduce alterations of the RPE barrier occurring during diabetic retinopathy.
Asunto(s)
Barrera Hematorretinal/fisiología , Edema Macular/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Proteínas Gestacionales/biosíntesis , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Animales , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , ADN/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Sustancias de Crecimiento , Humanos , Inmunohistoquímica , Inulina/farmacocinética , Edema Macular/patología , Epitelio Pigmentado Ocular/patología , Factor de Crecimiento Placentario , Proteínas Gestacionales/genética , Ratas , Ratas Endogámicas Lew , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
By the method of indirect immunohistochemistry, distribution of transferrin and of transferrin receptor of the type (TFR1) was studied in the formed rat eye retina at the period of early postnatal ontogenesis (from birth to opening of eyelids). It has been established that the character of distribution of these proteins and intensity of specific staining change dependent on the retina formation stage. Retina of the newborn rat is characterized by diffuse transferrin distribution in nuclear retina layers (in the neuroblast layer--NBL) and in the ganglionic cell layer (GCL) as well as in the eye pigment epithelium (PE); relative immunoreactivity to transferrin is not high. At the 5th postnatal day, immunoreactivity to transferrin is maximal and is revealed both in nuclear and in plexiform layers of retina and in the eye PE, the greatest signal being characteristic of NBL. At the 10th postnatal day the transferrin signal intensity in retina decreases, specific staining is revealed in GCL, PE, and in the area of formed outer segments of photoreceptors. At the 15th postnatal day, transferrin is revealed in GCL, in outer and inner photoreceptor segments and in the eye PE. TFR1 is present in all retinal layers at all stages of the retina formation; the relative immunoreactivity to TFR1 sharply rises beginning from the 10th postnatal day; correlation between distribution of transferrin and TFR1 is detected in the entire retina of newborn rats as well as in the external retina area at subsequent stages of its development. A possible role of transferrin at various stages of formation of retinal is discussed.
Asunto(s)
Receptores de Transferrina/metabolismo , Células Ganglionares de la Retina/metabolismo , Epitelio Pigmentado de la Retina/crecimiento & desarrollo , Epitelio Pigmentado de la Retina/metabolismo , Animales , Animales Recién Nacidos , Inmunohistoquímica , Ratas , Ratas Wistar , Células Ganglionares de la Retina/citología , Epitelio Pigmentado de la Retina/citología , Factores de TiempoRESUMEN
Pioneer work on iontophoresis undertaken by David Maurice during the 1970s and 1980s laid the initial groundwork for its potential implementation as a promising ocular therapeutic modality. A better understanding of tissue interactions within the eye during electric current application, along with better designs of drug delivery devices have enabled us to pursue David Maurice's original ideas and take them from the bench to the bed side. In the present study we demonstrate the potential application of an iontophoresis device (Eyegate, Optis, France) for the treatment of certain human eye diseases. Seventeen patients received a penetrating keratoplasty (PKP) at various intervals before presentation with active graft rejection in our clinic and were treated using this iontophoresis device. Methylprednisolone sodium succinate (MP) 62.5 mg/ml was infused within the Eyegate ocular probe container and an electrical current of 1.5 mA was delivered for 4 min with the negative pole connected to the ocular probe. Patients were treated on an ambulatory basis and received a standard course of three iontophoresis applications given once a day over 3 consecutive days. After treatment, 15 of the 17 treated eyes (88%) demonstrated a complete reversal of the rejection processes. In two eyes, only a partial and temporary improvement was observed. The mean best corrected visual acuity of all 17 patients during the last follow up visit was 0.37 +/- 0.2 compared to 0.06 +/- 0.05 before initiation of the iontophoresis treatment. The mean follow-up time was 13.7 months with a range of 5-29 months for the 17 patients. No significant side-effects associated with the iontophoresis treatment were observed. Thus, for the management of active corneal graft rejection, iontophoresis of MP can be an alternative to very frequent instillations of eye drops, or to pulsed intravenous therapy of corticosteroids.
Asunto(s)
Glucocorticoides/administración & dosificación , Rechazo de Injerto/tratamiento farmacológico , Iontoforesis/métodos , Queratoplastia Penetrante , Hemisuccinato de Metilprednisolona/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Sistemas de Liberación de Medicamentos/métodos , Estudios de Seguimiento , Glucocorticoides/uso terapéutico , Humanos , Hemisuccinato de Metilprednisolona/uso terapéutico , Persona de Mediana Edad , Resultado del Tratamiento , Agudeza VisualRESUMEN
We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the design of specific antiangiogenic strategy in diseases of the cornea.
Asunto(s)
Córnea/metabolismo , Técnicas de Transferencia de Gen , Iontoforesis/métodos , Oligonucleótidos Antisentido/química , Animales , Epitelio/metabolismo , Masculino , Microscopía Confocal , Neovascularización Fisiológica , Oligonucleótidos/química , Oligonucleótidos Antisentido/uso terapéutico , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
BACKGROUND: Intravitreal neovascular diseases, as in ischemic retinopathies, are a major cause of blindness. Because inflammatory mechanisms influence vitreal neovascularization and cyclooxygenase (COX)-2 promotes tumor angiogenesis, we investigated the role of COX-2 in ischemic proliferative retinopathy. METHODS AND RESULTS: We describe here that COX-2 is induced in retinal astrocytes in human diabetic retinopathy, in the murine and rat model of ischemic proliferative retinopathy in vivo, and in hypoxic astrocytes in vitro. Specific COX-2 but not COX-1 inhibitors prevented intravitreal neovascularization, whereas prostaglandin E2, mainly via its prostaglandin E receptor 3 (EP3), exacerbated neovascularization. COX-2 inhibition induced an upregulation of thrombospondin-1 and its CD36 receptor, consistent with the observed antiangiogenic effects of COX-2 inhibition; EP3 stimulation reversed effects of COX-2 inhibitors on thrombospondin-1 and CD36. CONCLUSIONS: These findings point to an important role for COX-2 in ischemic proliferative retinopathy, as in diabetes.
Asunto(s)
Retinopatía Diabética/enzimología , Isquemia/enzimología , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Receptores Inmunológicos , Vitreorretinopatía Proliferativa/enzimología , Adulto , Anciano , Animales , Astrocitos/efectos de los fármacos , Astrocitos/enzimología , Astrocitos/patología , Antígenos CD36/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Ciclooxigenasa 2 , Retinopatía Diabética/complicaciones , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/patología , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Factores de Crecimiento Endotelial/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Isquemia/complicaciones , Isquemia/patología , Isoenzimas/antagonistas & inhibidores , Linfocinas/metabolismo , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Ratas , Ratas Sprague-Dawley , Receptores de Lipoproteína/metabolismo , Receptores de Prostaglandina E/efectos de los fármacos , Receptores de Prostaglandina E/metabolismo , Subtipo EP3 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina E , Receptores Depuradores , Retina/efectos de los fármacos , Retina/enzimología , Retina/patología , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/patología , Trombospondina 1/metabolismo , Factor A de Crecimiento Endotelial Vascular , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial Vascular , Vitreorretinopatía Proliferativa/complicaciones , Vitreorretinopatía Proliferativa/tratamiento farmacológicoRESUMEN
L-DNase II is derived from its precursor leucocyte elastase inhibitor (LEI) by post-translational modification. In vitro, the conversion of LEI into L-DNase II can be induced by incubation of LEI at an acidic pH. In this study, we proposed to analyze the effects of intracellular acidification on this transformation. Amiloride derivatives, like hexamethylene amiloride (HMA), are known to provoke a decrease of cytosolic pH by inhibiting the Na(+)/H(+) antiport. In BHK cells, treatment with HMA-induced apoptosis accompanied by an increase in L-DNase II immunoreactivity and L-DNase II enzymatic activity. Overexpression of L-DNase II precursor led to a significant increase of apoptosis in these cells supporting the involvement of L-DNase II in HMA induced apoptosis. As previously shown in other cells, etoposide-induced apoptosis did not activate L-DNase. On the contrary, LEI overexpression significantly increased cell survival in etoposide-induced apoptosis. Together these results suggest differential roles of LEI and L-DNase II in response to different types of apoptotic inducers.
Asunto(s)
Amilorida/análogos & derivados , Apoptosis/fisiología , Endodesoxirribonucleasas/metabolismo , Serpinas/metabolismo , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Amilorida/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Cricetinae , Activación Enzimática/efectos de los fármacos , Etopósido/farmacología , Concentración de Iones de Hidrógeno , Serpinas/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Factores de TiempoRESUMEN
Transcorneoscleral iontophoresis was used to enhance ocular penetration of a 21-bp NH(2) protected anti-NOSII oligonucleotides (ODNs) (fluorescein or infrared-41 labeled) in Lewis rats. Both histochemical localization and acrylamide sequencing gels were used. To evaluate the potential to down-regulate NOSII expression in the rat model of endotoxin-induced uveitis (EIU), anti-sense NOSII ODN, scrambled ODN or saline were iontophorezed into these animals' eyes. Iontophoresis facilitated the penetration of intact ODNs into the intraocular tissues of the rat eye and only the eyes receiving ODNs and electrical current demonstrated intact ODNs within the ocular tissues of both segments of the eye. Iontophoresis of anti-NOSII ODN significantly down-regulated the expression of NOSII expression in iris/ciliary body compared to the saline or scrambled ODN treated eyes. Nitrite production was also significantly reduced in the anti-NOSII applied eyes compared to those treated with saline. Using this system, intraocular delivery of ODNs can be significantly enhanced increasing the potential for successful gene therapy for human eye diseases.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Endotoxinas/toxicidad , Óxido Nítrico Sintasa/genética , Oligonucleótidos Antisentido/farmacología , Uveítis/genética , Animales , Secuencia de Bases , Cartilla de ADN , Iontoforesis , Óxido Nítrico Sintasa de Tipo II , Ratas , Ratas Endogámicas Lew , Uveítis/inducido químicamente , Uveítis/enzimologíaRESUMEN
Retinal pigmented epithelium (RPE) cells are of central importance in the maintenance of neural retinal function. RPE cell apoptosis is responsible for the development of a variety of retinal degeneration. The role of FGF2 was investigated on RPE cell proliferation and apoptosis in vitro. In the absence of serum, RPE cells died by apoptosis, while the addition of FGF2 greatly reduces apoptosis over a 7-day culture period. This is due to an autocrine loop involving secretion of endogenous FGF1 in the mechanism that govern FGF2-induced resistance to apoptosis. FGF2 induces long-term activation of FGFR1 and ERK1/2, and production of the anti-apoptotic protein BcL-x. Because FGF1 has no classical signal sequence to direct its secretion, we investigated the effects of FGF1 secretion on RPE proliferation and apoptosis in the absence of exogenous FGF2. Forced secretion of endogenous FGF1 by adding a signal peptide to the FGF1 molecule induces FGF1 secretion and cell proliferation in the presence of serum, while in FGF1 stops to be secreted and cell die in the absence of serum. Conversely, in cells cultured in the presence of serum, FGF1 without signal peptide is not secreted, but is secreted and rescue RPE cell from apoptosis when cells are cultured without serum. Thus, the proliferation and survival activities of endogenous FGF1 depend on the secretion of FGF1 which is determined by the cell environment.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas del Ojo/fisiología , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Epitelio Pigmentado Ocular/efectos de los fármacos , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Adulto , División Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Activación Enzimática/efectos de los fármacos , Factor 1 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Epitelio Pigmentado Ocular/citología , Señales de Clasificación de Proteína/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesis , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Degeneración Retiniana/fisiopatología , Proteína bcl-XRESUMEN
In the retina, angiogenesis is an important component of normal physiological events such as embryonic vascular development. It is also involved in pathological processes including diabetic retinopathies and age-related macular degeneration, and tumour growth such as choroidal melanoma. Fibroblast growth factor (FGF) 2 and vascular endothelial cell growth factor (VEGF) are the two major angiogenic factors in the retina. We investigated the mechanism of proliferation and the regulation of the mitogenic properties of FGF2 and VEGF in cultures of chorocapillary endothelial cells (CEC). FGF2 is a strong mitogen for CEC and induced a 2.5-fold increase in cell proliferation after 4 days in culture in the absence of serum. In contrast, VEGF is a poor mitogen for CEC. FGF2, but not VEGF induces a large activation of MEK1, ERK1/2 and P90(RSK) during CEC proliferation. Pharmacological inhibition of Ras processing, and of MEK1 and ERK1/2 activation reduced only by 50% FGF2-induced cell proliferation, suggesting that there is another signalling pathway for CEC proliferation. Pharmacological inhibition of the PI 3-Kinase also inhibits by half FGF2-induced CEC proliferation. FGF2 stimulates the activation of the PI 3-K, P70(S6K) and Akt. Inhibition of both ERK1/2 and PI 3-K activities suppressed FGF2-induced CEC proliferation, demonstrating that CEC proliferation requires both ERKs and PI 3-K pathways. These data on the molecular mechanism and signalling may have important implications for providing more selective methods for anti-angiogenic and anti-tumoural therapy.
Asunto(s)
Capilares/fisiología , Coroides/irrigación sanguínea , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/fisiología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Linfocinas/farmacología , Animales , Capilares/citología , Bovinos , División Celular , Coroides/citología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Separación Inmunomagnética , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mitógenos/farmacología , Modelos Biológicos , Neovascularización Fisiológica , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor Cross-Talk , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
During retinal development, the neuronal death is carried out by the mechanism of apoptosis. Among the different endonucleases activated, L-DNase II seems to be responsible for most of DNA degradation in this tissue. L-DNase II derives from LEI (Leukocyte Elastase Inhibitor) by a post-translational modification carried out by elastase in apoptosis induced in vitro. In this study, we investigated whether elastase could be implicated in apoptosis occurring during retinal development. Although elastase and LEI/elastase complex are colocalized in retinal sections, the LEI/elastase complex, detected by Western blot, does not change at all stages of development. However, at pH 4 retinal extracts show an enhanced activation of the L-DNase II. These results suggest that an acid protease, such as a cathepsin, may be implicated in neuronal retinal apoptosis.
Asunto(s)
Apoptosis/fisiología , Endodesoxirribonucleasas/metabolismo , Elastasa Pancreática/metabolismo , Retina/metabolismo , Serpinas/metabolismo , Animales , Embrión de Pollo , Pollos , Activación Enzimática , Femenino , Retina/embriologíaRESUMEN
Intravitreal neovascular diseases are a major cause of blindness worldwide. It remains unclear why neovessels in many retinal diseases spread into the physiologically nonvascularized vitreous rather than into the ischemic retinal areas, where the angiogenic factors are released. Here we show that inducible nitric oxide synthase (iNOS) is expressed in the ischemic retina. Using iNOS knockout mice and the iNOS inhibitor 1400W, we demonstrate that iNOS expression inhibits angiogenesis locally in the avascular retina, mediated at least in part by a downregulation of VEGF receptor 2 (VEGFR2) in cells adjacent to iNOS-expressing cells. At the same time, pathological intravitreal neovascularization is considerably stronger in iNOS-expressing animals. These findings demonstrate that iNOS plays a crucial role in retinal neovascular disease and show that it offers an ideal target for the control of vitreal neovascularization through improvement of the vascularization of the hypoxic retina.
Asunto(s)
Isquemia/enzimología , Neovascularización Patológica , Óxido Nítrico Sintasa/fisiología , Retina/enzimología , Vasos Retinianos/enzimología , Cuerpo Vítreo/irrigación sanguínea , Cuerpo Vítreo/enzimología , Animales , Secuencia de Bases , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Humanos , Isquemia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa/deficiencia , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retina/patología , Vasos Retinianos/patologíaRESUMEN
During the development of the neural retina, 50% of the neurons die physiologically by apoptosis. In the chick embryo, the apoptotic wave starts at E8 and ends at E18, with a peak at E11. The onset of apoptosis is accompanied by the activation of several degradative enzymes. Among these, the activation of the endonucleases leads to the degradation of the genomic DNA of the cell which is thought to be the final event in apoptosis. Here, we have investigated the endonucleases activated during apoptosis associated with retinal development. We have found that Ca2+-Mg2+-dependent endonucleases, as well as acid endonucleases are activated. The results obtained in vitro using purified nuclei from chicken retina indicate that the endonuclease activity resulting from the activation of L-DNase II, an acid DNase is responsible for most of the DNA degradation observed in these cells.
Asunto(s)
Apoptosis/fisiología , Pollos/crecimiento & desarrollo , Endodesoxirribonucleasas/fisiología , Retina/fisiología , Animales , Núcleo Celular/genética , Núcleo Celular/patología , Electroforesis en Gel de Poliacrilamida , Retina/patologíaRESUMEN
PURPOSE: To analyze the intracellular signaling involved in the proliferation of choroidal endothelial cells (CECs) in vitro. METHODS: Bovine CECs were cultured in endothelial growth medium (EGM) containing 2% fetal calf serum (FCS), 10 microg/ml bovine brain extract (BBE), and 10 ng/ml epidermal growth factor (EGF) in fibronectin-coated plates. Cells were treated with various specific pharmacologic inhibitors of the mitogen-activated protein kinase (MAPK) and of the phosphatidylinositol 3-kinase (PI 3-K) pathways to analyze signaling involved in CEC proliferation. Activation of the MAPK and PI 3-K was detected by Western blot analysis, using specific antiphosphosignaling protein antibodies. RESULTS: FCS, EGF, and BBE were all necessary to induce optimal CEC proliferation. Individually, these three components were not mitogenic. EGM-stimulated CEC proliferation involved the activation of the Raf/mitogen extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90(RSK) cascade. Inhibition of Ras resulted in a 92% reduction of CEC proliferation, whereas inhibition of ERK1/2 activity reduced it by only 46%. The PI 3-K/p70(S6K)/Akt pathway was also stimulated during CEC proliferation, and inhibition of PI 3-K activity resulted in a 94% reduction in CEC proliferation. Inhibition of PI 3-K/p70(S6K) activities also unexpectedly inhibited ERK activity, whereas the converse was not observed, suggesting that PI 3-K acted upstream from ERK and controlled this pathway for CEC proliferation. CONCLUSIONS: CEC proliferation involves both ERK and PI 3-K. That PI 3-K signaling is a key component in cell proliferation can be demonstrated by controlling ERK activity. These data on the molecular mechanism and signaling of CEC proliferation may have major implications for developing more selective methods for antiangiogenic and antitumoral therapy.
Asunto(s)
Coroides/irrigación sanguínea , Endotelio Vascular/citología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Animales , Western Blotting , Bovinos , División Celular/fisiología , Células Cultivadas , Endotelio Vascular/enzimología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-aktRESUMEN
Fibroblast growth factor 1 (FGF1) induces proliferation and differentiation in a wide variety of cells of mesodermal and neuroectodermal origin. FGF1 has no 'classical' signal sequence to direct its secretion, and there has been considerable debate concerning FGF1 secretion and its role in the biological activities of FGF1. We investigated the effects of FGF1 secretion and the signalling induced by signal peptide (SP)-containing FGFI and SP-less FGF1, on the proliferation and the apoptosis in retinal pigmented epithelial (RPE) cells. Primary RPE cell cultures were transfected with FGF1 (FGF1 cells) and SP-FGF1 (SP-FGF1 cells) cDNAs. SP-FGF1 cells secreted large amount of FGF1 and actively proliferated, whereas FGF1 and control cells did not. Secreted FGF1 induced short-term activation of both FGFR1 and ERK2, which were required for cell proliferation. In contrast, SP-FGF1 cells stopped secreting FGF1 and died rapidly, if cultured in the absence of serum. Surprisingly, FGF1 cells, but not control cells, secreted FGF1 and were resistant to apoptosis induced by serum depletion. Secreted FGF1 induced long-term activation of FGFR1 and ERK2, which was necessary to induce a constant and high level of Bcl-x production, and to induce cell survival in FGFI cells. Downregulation of ERK2 and Bcl-x increased apoptosis. Thus, the proliferation and survival activities of FGF1 depend on the secretion of FGF1 which is determined by the cell culture conditions. Cell proliferation was SP-dependent, whereas cell survival was not. The signal peptide controls the level and duration, 'whispering or shouting', of ERK2 activation cells which determines FGF1 biological function and may have important implications for anti-degenerative and anti-proliferative treatments.
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Proteínas del Ojo/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Epitelio Pigmentado Ocular/citología , Animales , Apoptosis/efectos de los fármacos , Bovinos , División Celular , Supervivencia Celular , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , ADN Complementario/genética , Activación Enzimática , Factor 1 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Fosforilación , Epitelio Pigmentado Ocular/metabolismo , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/biosíntesis , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal , Transfección , Proteína bcl-XRESUMEN
Leukocyte elastase inhibitor (LEI) is a cytosolic component of lung macrophages and blood leukocytes that inhibits neutrophil elastase. LEI is a member of the serpin superfamily, these proteins, mostly protease inhibitors, are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the hinge region of the reactive centre loop into a beta-sheet of the inhibitor. In this work three mutations were produced in the hinge region of elastase inhibitor that abolish the inhibition activity of LEI and transform the protein in a substrate of the elastase. This result demonstrates that the inhibitory mechanism of serpin is common to LEI.
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Serpinas/genética , Serpinas/farmacología , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
PURPOSE: The retina and other tissues need iron to survive. However, the normal iron metabolism in rodent retinas had not been characterized. This study was intended to investigate iron and iron homeostasis protein (ferritin, transferrin [Tf] and transferrin receptor [Tf-R]) distribution in 20- to 55-day-old rat retinas. METHODS: Iron was revealed on retinal sections directly by proton-induced x-ray emission (PIXE) and indirectly by electron microscopy (EM). Ferritin, Tf, and Tf-R proteins were localized by immunohistochemistry. Transferrin expression was localized by in situ hybridization (ISH). Transferrin and ferritin proteins and mRNA were analyzed by Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR), respectively. RESULTS: Iron is widely and unevenly distributed throughout the adult rat retina. The highest concentration was observed by PIXE in the choroid and the retinal pigmented epithelial cell (RPE) layer, and in inner segments of photoreceptors (IS). Outer segments of photoreceptors (OS) also contain iron. EM studies suggested the presence of iron inclusions inside the photoreceptor discs. Choroid, RPE, and IS showed a strong immunoreactivity for ferritin. Transferrin accumulated mainly in the IS and OS areas and in RPE cells but can also be detected slightly in retinal capillaries. Western blot analysis for Tf and ferritin confirmed their presence in the adult neural retina. By RT-PCR, H- and L-chains of ferritin and Tf mRNAs were expressed in neural retina, but the main sites of Tf synthesis observed by ISH were the RPE and choroid cell layers. Tf-R immunoreactivity was detected in the ganglion cell layer, inner nuclear layer, outer plexiform layer, IS, RPE, and choroid. These results were similar for all stages studied. CONCLUSIONS: For the first time, the present study characterized both iron and iron homeostasis proteins in rodent retinas. In the outer retina, iron and ferritin shared the same distribution patterns. In contrast, Tf, mainly synthesized by RPE cells and detected in OS and IS areas, probably helps to transport iron to photoreceptors through their Tf-R. This is a likely pathway for filling iron needs in the outer retina.
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Ferritinas/metabolismo , Hierro/metabolismo , Receptores de Transferrina/metabolismo , Retina/metabolismo , Transferrina/metabolismo , Animales , Western Blotting , Sondas de ADN/química , Microanálisis por Sonda Electrónica , Ferritinas/genética , Técnica del Anticuerpo Fluorescente Indirecta , Hibridación in Situ , ARN Mensajero/biosíntesis , Radioinmunoensayo , Ratas , Ratas Long-Evans , Ratas Mutantes , Ratas Wistar , Retina/ultraestructura , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transferrina/genéticaRESUMEN
Fibroblast growth factor (FGF) 2 is a survival factor for various cell types, including retinal neurons. However, little is understood about the molecular bases of the neuroprotective role of FGF2 in the retina. In this report, FGF2 survival activity was studied in chick retinal neurons subjected to apoptosis by serum deprivation. Exogenous FGF2 supported neuronal survival after serum deprivation and increased neuronal bcl-x(L) and bcl-2 expression, through binding to its receptor R1 (FGF-R1), and subsequent extracellular signal-regulated kinase (ERK) activation. Endogenous FGF2 was transiently overexpressed after serum deprivation. Its down-regulation by antisense oligonucleotides and blockade of its signaling pathway (binding to FGF-R1, tyrosine phosphorylation, and ERK inhibition) decreased bcl-x(L) and bcl-2 levels and and enhanced apoptosis, suggesting that endogenous FGF2 supported neuronal survival through a pathway similar to that of exogenous FGF2. This pathway may serve to up-regulate, or maintain, bcl-x(L) and bcl-2 levels that normally decrease during the onset of apoptosis. Indeed, long-term ERK activation and high bcl-x(L) levels are necessary for the survival activity of both exogenous and endogenous FGF2. Because FGF2 is upregulated following retinal injury in vivo, we suggest that an injury-stimulated autocrine/paracrine FGF2 loop may serve to maintain high levels of survival proteins, such as Bcl-x(L), through ERK activation in retinal neurons.
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Supervivencia Celular/fisiología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Neuronas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Retina/embriología , Animales , Apoptosis , Embrión de Pollo , Medio de Cultivo Libre de Suero , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Transducción de Señal , Proteína bcl-XRESUMEN
The metabolic conversion of n-3 fatty acids was studied in the human Y79 retinoblastoma cell line. Cultured cells were exposed to increasing concentrations of either 18:3n-3, 22:5n-3, or 22:6n-3, and their phospholipid fatty acid composition was analyzed after 72 hr. Cells internalized the supplemental fatty acids and proceeded to their metabolic conversion. Supplemental 22:6n-3 was directly esterified into cell phospholipids, at levels typical for normal neural retinas (41% by weight of phosphatidylethanolamine fatty acids, and 24% of phosphatidylcholine fatty acids). In contrast, 18:3n-3 was mainly converted to 20:5n-3 and 22:5n-3, both of which appeared in cell phospholipids after exposure to low external concentrations of 18:3n-3 (10 microg/ml). Y79 cells can proceed to the metabolic conversion of 18:3n-3 through elongation and Delta6- and Delta5-desaturation. When cells were exposed to high external concentrations of 18:3n-3 (30 microg/ml), the supplemental fatty acid was directly incorporated, and its relative content increased in both phospholipid classes to the detriment of all other n-3 fatty acids. Cells cultured in the presence of 22:5n-3 did not incorporate 22:6n-3 into their phospholipids but did incorporate 20:5n-3 and 22:5n-3. The data suggest that Y79 cells can proceed to the microsomal steps of n-3 metabolism, involving elongation, desaturation, and chain shortening of 22C fatty acids. Although Y79 cells avidly used supplemental 22:6n-3 for phospholipid incorporation at levels typical for normal photoreceptor cells, they failed to match such levels through metabolic conversion of n-3 parent fatty acids. The terminal step of the very long-chain polyunsaturated fatty acid synthesis, consisting in Delta6-desaturation followed by peroxisomal chain shortening of 24C-fatty acids, could be rate-limiting in Y79 cells.