RESUMEN
Hydrocinnamoyl-L-valylpyrrolidine (AS-1), a synthetic low-molecule mimetic of myeloid differentiation primary response gene 88 (MyD88), inhibits inflammation by disrupting the interaction between the interleukin-1 receptor (IL-1R) and MyD88. Here, we describe the effects of AS-1 on injury-induced increases in inflammation and neovascularization in mouse corneas. Mice were administered a subconjunctival injection of 8 µL AS-1 diluent before or after corneal alkali burn, followed by evaluation of corneal resurfacing and corneal neovascularization (CNV) by slit-lamp biomicroscopy and clinical assessment. Corneal inflammation was assessed by whole-mount CD45+ immunofluorescence staining, and corneal hemangiogenesis and lymphangiogenesis following injury were evaluated by immunostaining for the vascular markers isolectin B4 (IB4) and the lymphatic vascularized marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), respectively. Additionally, corneal tissues were collected to determine the expression of 35 cytokines, and we detected activation of IL-1RI, MyD88, and mitogen-activated protein kinase (MAPK). The results showed that alkali conditions increased the number of CD45+ cells and expression of vascular endothelial growth factor (VEGF)-A, VEGF-C, and LYVE1 in corneas, with these levels decreased in the AS-1-treated group. Moreover, AS-1 effectively prevented alkali-induced cytokine production, blocked interactions between IL-1RI and MyD88, and inhibited MAPK activation post-alkali burn. These results indicated that AS-1 prevented alkali-induced corneal hemangiogenesis and lymphangiogenesis by blocking IL-1RI-MyD88 interaction, as well as extracellular signal-regulated kinase phosphorylation, and could be efficacious for the prevention and treatment of corneal alkali burn.
Asunto(s)
Quemaduras Químicas/prevención & control , Neovascularización de la Córnea/prevención & control , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quemaduras Oculares/inducido químicamente , Pirrolidinas/uso terapéutico , Valina/análogos & derivados , Inhibidores de la Angiogénesis , Animales , Biomarcadores/metabolismo , Western Blotting , Quemaduras Químicas/enzimología , Quemaduras Químicas/patología , Neovascularización de la Córnea/enzimología , Neovascularización de la Córnea/patología , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quemaduras Oculares/enzimología , Quemaduras Oculares/patología , Proteínas del Ojo/metabolismo , Humanos , Inmunoprecipitación , Linfangiogénesis/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Fosforilación , Reacción en Cadena en Tiempo Real de la Polimerasa , Hidróxido de Sodio , Valina/uso terapéuticoRESUMEN
PURPOSE: To explore whether alkali burn causes corneal neuropathic pain and activates the neuropathic pain matrix in the central nervous system in mice. METHODS: A corneal alkali burn mouse model (grade II) was used. The mechanical threshold in the cauterized area was tested using Von Frey hairs. Spontaneous pain behavior was investigated with conditioned place preference. Phosphor extracellular signal-regulated kinase (ERK), which is a marker for neuronal activation in chronic pain processing, was investigated in several representative areas of the neuropathic pain matrix: the 2 regions of the spinal trigeminal nucleus (subnucleus interpolaris/caudalis, Vi/Vc; subnucleus caudalis/upper cervical cord, Vc/C1), insular cortex, anterior cingulated cortex (ACC), and the rostroventral medulla (RVM). Furthermore, pharmacologically blocking pERK activation in the ACC of alkali burn mice was performed in a separate study. RESULTS: Corneal alkali burn caused long-lasting damage to the corneal subbasal nerve fibers, and mice exhibited spontaneous pain behavior. By testing in several representative areas of the neuropathic pain matrix in the higher nervous system, phosphor ERK was significantly activated in Vc/C1, but not in Vi/Vc. Also, ERK was activated in the insular cortex, ACC, and RVM. Furthermore, pharmacologically blocking ERK activation in the ACC abolished alkali burn induced corneal spontaneous pain. CONCLUSIONS: Alkali burn could cause corneal spontaneous pain and activate the neuropathic pain matrix in the central nervous system. Furthermore, activation of ERK in the ACC is required for alkali burn induced corneal spontaneous pain.
Asunto(s)
Quemaduras Químicas/fisiopatología , Enfermedades del Sistema Nervioso Central/fisiopatología , Enfermedades de la Córnea/fisiopatología , Quemaduras Oculares/inducido químicamente , Dolor Ocular/fisiopatología , Neuralgia/fisiopatología , Hidróxido de Sodio/toxicidad , Animales , Quemaduras Químicas/enzimología , Cáusticos/toxicidad , Enfermedades del Sistema Nervioso Central/enzimología , Córnea/inervación , Enfermedades de la Córnea/enzimología , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quemaduras Oculares/enzimología , Quemaduras Oculares/fisiopatología , Dolor Ocular/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuralgia/enzimología , Dimensión del Dolor , Umbral del Dolor , FosforilaciónRESUMEN
Citrullination is an important posttranslational modification that occurs during retinal gliosis. We examined the expression of peptidyl arginine deiminases (PADs) to identify the PADs that mediate citrullination in a model of alkali-induced retinal gliosis. Mouse corneas were exposed to 1.0 N NaOH and posterior eye tissue from injured and control uninjured eyes was evaluated for transcript levels of various PADs by reverse-transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR (qPCR). Retinas were also subjected to immunohistochemistry (IHC) for glial fibrillary acidic protein (GFAP), citrullinated species, PAD2, and PAD4 and tissue levels of GFAP, citrullinated species, and PAD4 were measured by western blots. In other experiments, the PAD4 inhibitor streptonigrin was injected intravitreally into injured eyes ex vivo to test inhibitory activity in an organ culture system. We found that uninjured retina and choroid expressed Pad2 and Pad4 transcripts. Pad4 transcript levels increased by day 7 post-injury (p < 0.05), whereas Pad2 levels did not change significantly (p > 0.05) by qPCR. By IHC, PAD2 was expressed in uninjured eyes along ganglion cell astrocytes, but in injured retina PAD2 was downregulated at 7 days. On the other hand, PAD4 showed increased staining in the retina upon injury revealing a pattern that overlapped with filamentous GFAP staining in Müller glial processes by 7 days. Injury-induced citrullination and soluble GFAP protein levels were reduced by PAD4 inhibition in western blot experiments of organ cultures. Together, our findings for the first time identify PAD4 as a novel injury-inducible druggable target for retinal gliosis.
Asunto(s)
Quemaduras Oculares/enzimología , Gliosis/enzimología , Hidrolasas/metabolismo , Retina/enzimología , Retina/lesiones , Enfermedades de la Retina/enzimología , Animales , Arginina/metabolismo , Quemaduras Químicas/enzimología , Citrulina/metabolismo , Quemaduras Oculares/inducido químicamente , Quemaduras Oculares/complicaciones , Femenino , Gliosis/inducido químicamente , Masculino , Ratones , Arginina Deiminasa Proteína-Tipo 4 , Enfermedades de la Retina/inducido químicamente , Enfermedades de la Retina/etiología , Hidróxido de SodioRESUMEN
Chemical burns are a major cause of corneal injury. Oxidative stress, inflammatory responses and neovascularization after the chemical burn aggravate corneal damage, and lead to loss of vision. Although NADPH oxidases (Noxs) play a crucial role in the production of reactive oxygen species (ROS), the role of Noxs in chemical burn-induced corneal injury remains to be elucidated. In the present study, the transcription and expression of Noxs in corneas were examined by RT-qPCR, western blot analysis and immunofluorescence staining. It was found that alkali burns markedly upregulated the transcription and expression of Nox2 and Nox4 in human or mouse corneas. The inhibition of Noxs by diphenyleneiodonium (DPI) or apocynin (Apo) effectively attenuated alkali burn-induced ROS production and decreased 3-nitrotyrosine (3-NT) protein levels in the corneas. In addition, Noxs/CD11b doubleimmunofluorescence staining indicated that Nox2 and Nox4 were partially co-localized with CD11b. DPI or Apo prevented the infiltration of CD11b-positive inflammatory cells, and inhibited the transcription of inflammatory cytokines following alkali burn-induced corneal injury. In our mouse model of alkali burn-induced corneal injury, corneal neovascularization (CNV) occurred on day 3, and it affected 50% of the whole area of the cornea on day 7, and on day 14, CNV coverage of the cornea reached maximum levels. DPI or Apo effectively attenuated alkali burninduced CNV and decreased the mRNA levels of angiogenic factors, including vascular endothelial growth factor (VEGF), VEGF receptors and matrix metalloproteinases (MMPs). Taken together, our data indicate that Noxs play a role in alkali burn-induced corneal injury by regulating oxidative stress, inflammatory responses and CNV, and we thus suggest that Noxs are a potential therapeutic target in the future treatment of chemical-induced corneal injury.
Asunto(s)
Quemaduras Químicas/enzimología , Lesiones de la Cornea/enzimología , Quemaduras Oculares/enzimología , NADPH Oxidasas/metabolismo , Acetofenonas/farmacología , Álcalis , Animales , Quemaduras Químicas/complicaciones , Quemaduras Químicas/tratamiento farmacológico , Quemaduras Químicas/patología , Lesiones de la Cornea/complicaciones , Lesiones de la Cornea/tratamiento farmacológico , Lesiones de la Cornea/patología , Neovascularización de la Córnea/complicaciones , Neovascularización de la Córnea/tratamiento farmacológico , Neovascularización de la Córnea/enzimología , Neovascularización de la Córnea/patología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Quemaduras Oculares/complicaciones , Quemaduras Oculares/tratamiento farmacológico , Quemaduras Oculares/patología , Humanos , Inflamación/patología , Ratones Endogámicos C57BL , Compuestos Onio/farmacología , Estrés Oxidativo/efectos de los fármacosRESUMEN
Our previous studies have shown that Dexamethasone (Dex) reduced the expression of matrix-metalloproteinases (MMPs -1,-3,-9,-13), IL-1ß and IL-6, while it significantly increased MMP-8 mRNA transcripts in a concomitant dry eye and corneal alkali burn murine model (CM). To investigate if MMP-8 induction is responsible for some of the protective effects of Dex in CM, MMP-8 knock out mice (MMP-8KO) were subjected to the CM for 2 or 5 days and topically treated either with 2 µl of 0.1% Dexamethasone (Dex), or saline QID. A separate group of C57BL/6 mice were topically treated with Dex or BSS and received either 100 nM CAM12 (MMP-8 inhibitor) or vehicle IP, QD. Here we demonstrate that topical Dex treated MMP-8KO mice subjected to CM showed reduced corneal clarity, increased expression of inflammatory mediators (IL-6, CXCL1, and MMP-1 mRNA) and increased neutrophil infiltration at 2D and 5D compared to Dex treated WT mice. C57BL/6 mice topically treated with Dex and CAM12 IP recapitulated findings seen with MMP-8KO mice. These results suggest that some of the anti-inflammatory effects of Dex are mediated through increased MMP-8 expression. J. Cell. Physiol. 231: 2506-2516, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Quemaduras Químicas/tratamiento farmacológico , Quemaduras Químicas/enzimología , Córnea/patología , Dexametasona/uso terapéutico , Síndromes de Ojo Seco/complicaciones , Quemaduras Oculares/tratamiento farmacológico , Quemaduras Oculares/enzimología , Metaloproteinasa 8 de la Matriz/metabolismo , Álcalis , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Quemaduras Químicas/complicaciones , Córnea/enzimología , Desecación , Dexametasona/farmacología , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/enzimología , Quemaduras Oculares/complicaciones , Femenino , Metaloproteinasa 8 de la Matriz/deficiencia , Metaloproteinasa 8 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Inhibidores de la Metaloproteinasa de la Matriz/uso terapéutico , Ratones Endogámicos C57BL , Ratones Noqueados , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés FisiológicoRESUMEN
PURPOSE: To evaluate the effects of dry eye on ocular surface protease activity and sight threatening corneal complications following ocular surface chemical injury. METHODS: C57BL/6 mice were subjected to unilateral alkali burn (AB) with or without concomitant dry eye for 2 or 5 days. Mice were observed daily for appearance of corneal perforation. Whole corneas were harvested and lysed for RNA extraction. Quantitative real-time PCR was performed to measure expression of inflammation cytokines, matrix metalloproteinases (MMP). Matrix metalloproteinase-9 activity, gelatinase activity, and myeloperoxidase (MPO) activity were evaluated in corneal lysates. Presence of infiltrating neutrophils was evaluated by immunohistochemistry and flow cytometry. RESULTS: Eyes subjected to the combined model of AB and dry eye (CM) had 20% sterile corneal perforation rate as soon as 1 day after the initial injury, which increased to 35% by 5 days, delayed wound closure and increased corneal opacity. Increased levels of IL-1ß, -6, and MMPs-1, -3, -8, -9, and -13, and chemokine (C-X-C motif) ligand 1 (CSCL1) transcripts were found after 2 days in CM compared with AB corneas. Increased MMP-1, -3, -9, and -13 immunoreactivity and gelatinolytic activity were seen in CM corneas compared with AB. Increased neutrophil infiltration and MPO activity was noted in the CM group compared with AB 2 days post injury. CONCLUSIONS: Desiccating stress worsens outcome of ocular AB, creating a cytokine and protease storm with greater neutrophil infiltration, increasing the risk of corneal perforation.
Asunto(s)
Quemaduras Químicas/genética , Quemaduras Oculares/genética , Regulación de la Expresión Génica , Metaloproteinasa 9 de la Matriz/genética , Estrés Oxidativo , ARN/genética , Cicatrización de Heridas , Álcalis/toxicidad , Animales , Quemaduras Químicas/enzimología , Quemaduras Químicas/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Quemaduras Oculares/inducido químicamente , Quemaduras Oculares/enzimología , Quemaduras Oculares/patología , Citometría de Flujo , Metaloproteinasa 9 de la Matriz/biosíntesis , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
PURPOSE: To investigate the effects and mechanisms of fasudil hydrochloride (fasudil) on and in alkali burn-induced corneal neovascularization (CNV) in mice. METHODS: To observe the effect of fasudil, mice with alkali-burned corneas were treated with either fasudil eye drops or phosphate-buffered saline (PBS) four times per day for 14 consecutive days. After injury, CNV and corneal epithelial defects were measured. The production of reactive oxygen species (ROS) and heme oxygenase-1(HO-1) was measured. The infiltration of polymorphonuclear neutrophils (PMNs) and the mRNA expressions of CNV-related genes were analyzed on day 14. RESULTS: The incidence of CNV was significantly lower after treatment with 100 µM and 300 µM fasudil than with PBS, especially with 100 µM fasudil. Meanwhile, the incidences of corneal epithelial defects was lower (n=15, all p<0.01). After treatment with 100 µM fasudil, the intensity of DHE ï¬uorescence was reduced in the corneal epithelium and stroma than with PBS treatment (n=5, all p<0.01), and the number of filtrated PMNs decreased. There were significant differences between the expressions of VEGF, TNF-a, MMP-8, and MMP-9 in the 100 µM fasudil group and the PBS group (n=8, all p<0.05). The production of HO-1 protein in the 100 µM fasudil group was 1.52±0.34 times more than in the PBS group (n=5 sample, p<0.05). CONCLUSIONS: 100 µM fasudil eye drops administered four times daily can significantly inhibit alkali burn-induced CNV and promote the healing of corneal epithelial defects in mice. These effects are attributed to a decrease in inflammatory cell infiltration, reduction of ROS, and upregulation of HO-1 protein after fasudil treatment.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Quemaduras Químicas/tratamiento farmacológico , Quemaduras Químicas/enzimología , Neovascularización de la Córnea/prevención & control , Quemaduras Oculares/tratamiento farmacológico , Quemaduras Oculares/enzimología , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Álcalis/toxicidad , Animales , Quemaduras Químicas/patología , Neovascularización de la Córnea/etiología , Neovascularización de la Córnea/patología , Modelos Animales de Enfermedad , Quemaduras Oculares/patología , Femenino , Hemo-Oxigenasa 1/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
PURPOSE: To investigate the effect of AMA0526, a specific inhibitor of rho-associated protein kinase (ROCK), on corneal neovascularization (NV) and scarring in different in vitro and in vivo experimental models. METHODS: The effect of AMA0526 on cell viability, proliferation, and migration of human umbilical vein endothelial cells was determined. Its in vivo topical effect on NV was investigated in the corneal micropocket mouse model (bevacizumab as a control). The vessel length, clock hours, and NV area were measured on photographs. The effect of AMA0526 on pathological wound healing was investigated in the alkali burn mouse model (dexamethasone as a control). Corneas were scored for corneal opacity (CO) and NV after burn injury. Immunohistochemistry was performed to study inflammation, blood vessel density, and collagen III deposition after 7 days. RESULTS: ROCK inhibition significantly inhibited vascular endothelial cell proliferation and migration in vitro in a dose-dependent manner. In the micropocket model, NV was significantly reduced by AMA0526 (37% reduction, P < 0.05) comparable to bevacizumab. CO and NV were reduced after AMA0526, compared with the vehicle (P < 0.05 at all time points from day 3) after chemical burn. AMA0526 resulted in decreased inflammatory cell infiltration (26% reduction, P < 0.01), angiogenesis (47% reduction, P < 0.01), and collagen III deposition (27% reduction, P = 0.009) in the alkali burn model. AMA0526 administration showed results similar to those of dexamethasone with an additional antifibrotic effect. CONCLUSIONS: The ROCK inhibitor, AMA0526, efficiently inhibited angiogenesis in vitro, reduced CO and NV, and controlled the complete process of wound healing in vivo. These results warrant further investigation of the therapeutic potential of AMA0526 for corneal NV and scarring.
Asunto(s)
Quemaduras Químicas/prevención & control , Opacidad de la Córnea/prevención & control , Inhibidores Enzimáticos/farmacología , Quemaduras Oculares/inducido químicamente , Neovascularización Patológica/prevención & control , Inhibidores de Proteínas Quinasas/farmacología , Cicatrización de Heridas/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidores , Animales , Quemaduras Químicas/enzimología , Quemaduras Químicas/etiología , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Colágeno Tipo III/metabolismo , Opacidad de la Córnea/inducido químicamente , Opacidad de la Córnea/enzimología , Dexametasona/farmacología , Modelos Animales de Enfermedad , Combinación de Medicamentos , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Quemaduras Oculares/enzimología , Glucocorticoides/farmacología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/enzimología , Neovascularización Patológica/etiología , Hidróxido de SodioRESUMEN
PURPOSE: Sulfur mustard (SM) induces acute ocular lesions, including erosions and inflammation that may be followed by delayed injuries expressed by epithelial defects and neovascularization (NV). Based on the matrix metalloproteinases (MMPs) activity, we evaluated the clinical and biochemical effects of topical treatment with doxycycline, an MMP inhibitor, targeted to the various injury stages. METHODS: Rabbit eyes were exposed to SM vapor. A clinical follow-up was carried out up to 2 months. Tear fluid and cornea samples were collected at different time points for measurements of MMPs activity by zymography. Efficacy of a post-exposure topical doxycycline (2 mg/ml in phosphate buffer saline, ×4/d), targeted to the different phases of the clinical injury, was evaluated. RESULTS: Elevated MMP-9 and MMP-2 activities were found in all corneas during the acute injury and in vascularized corneas during the delayed pathology. In the tear fluid, high MMP-9 activity and negligible MMP-2 activity were found in all the exposed eyes until after the appearance of the delayed pathology symptoms. Prolonged doxycycline treatment reduced MMP-9 activity in the tear fluid. During the acute phase, doxycycline treatment reduced corneal MMP-9 activity and the severity of the injury. Targeting the delayed pathology, doxycycline was clinically efficient only when treatment began before NV appearance. CONCLUSIONS: This in vivo study showed the involvement of MMP-9 and MMP-2 during different phases of the SM-induced ocular injury, and the potential of doxycycline treatment as a post exposure measure for reducing the acute injury and as a preventive therapy for ameliorating the delayed pathology. The tear fluid provided a non-invasive method for continuous follow-up of MMPs activity and revealed additional beneficial aspects of injury and the treatment.
Asunto(s)
Quemaduras Químicas/tratamiento farmacológico , Lesiones de la Cornea/tratamiento farmacológico , Doxiciclina/uso terapéutico , Quemaduras Oculares/tratamiento farmacológico , Metaloproteinasas de la Matriz/metabolismo , Animales , Antibacterianos/administración & dosificación , Antibacterianos/uso terapéutico , Quemaduras Químicas/enzimología , Quemaduras Químicas/patología , Lesiones de la Cornea/inducido químicamente , Lesiones de la Cornea/enzimología , Modelos Animales de Enfermedad , Doxiciclina/administración & dosificación , Quemaduras Oculares/enzimología , Quemaduras Oculares/patología , Femenino , Inhibidores de la Metaloproteinasa de la Matriz/uso terapéutico , Metaloproteinasas de la Matriz/efectos de los fármacos , Gas Mostaza/toxicidad , Soluciones Oftálmicas , Conejos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Neovascularization (NV) in the cornea is a major cause of vision impairment and corneal blindness. Hemangiogenesis and lymphangiogenesis induced by inflammation underlie the pathogenesis of corneal NV. The current mainstay treatment, corticosteroid, treats the inflammation associated with corneal NV, but is not satisfactory due to such side effects as cataract and the increase in intraocular pressure. It is imperative to develop a novel therapy that specifically targets the hemangiogenesis, lymphangiogenesis, and inflammation pathways underlying corneal NV. Histone deacetylase inhibitors (HDACi) have been in clinical trials for cancer and other diseases. In particular, HDACi suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) has been approved by the FDA for the treatment of cutaneous T-cell lymphoma. The functional mechanism of SAHA in cancer and especially in corneal NV remains unclear. Here, we show that topical application of SAHA inhibits neovascularization in an alkali-burn corneal injury model. Mechanistically, SAHA inhibits corneal NV by repressing hemangiogenesis, inflammation pathways, and previously overlooked lymphangiogenesis. Topical SAHA is well tolerated on the ocular surface. In addition, the potency of SAHA in corneal NV appears to be comparable to the current steroid therapy. SAHA may possess promising therapeutic potential in alkali-burn corneal injury and other inflammatory neovascularization disorders.
Asunto(s)
Quemaduras Químicas/tratamiento farmacológico , Enfermedades de la Córnea/tratamiento farmacológico , Lesiones de la Cornea , Quemaduras Oculares/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Álcalis , Animales , Quemaduras Químicas/patología , Córnea/irrigación sanguínea , Córnea/efectos de los fármacos , Córnea/enzimología , Enfermedades de la Córnea/patología , Neovascularización de la Córnea/inducido químicamente , Neovascularización de la Córnea/tratamiento farmacológico , Neovascularización de la Córnea/patología , Modelos Animales de Enfermedad , Quemaduras Oculares/inducido químicamente , Quemaduras Oculares/enzimología , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Inflamación/tratamiento farmacológico , Inflamación/patología , Linfangiogénesis/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , VorinostatRESUMEN
Pigment epithelium-derived factor (PEDF) is an intrinsic antiangiogenic factor and a potential therapeutic agent. Previously, we discovered the mechanism of PEDF-induced apoptosis of human umbilical vein endothelial cells (HUVECs) as sequential induction/activation of p38 mitogen-activated protein kinase (MAPK), peroxisome proliferator-activated receptor gamma (PPAR-gamma), and p53. In the present study, we investigated the signaling role of cytosolic calcium-dependent phospholipase A(2)-alpha (cPLA(2)-alpha) to bridge p38 MAPK and PPAR-gamma activation. PEDF induced cPLA(2)-alpha activation in HUVECs and in endothelial cells in chemical burn-induced vessels on mouse cornea. The cPLA(2)-alpha activation is evident from the phosphorylation and nuclear translocation of cPLA(2)-alpha as well as arachidonic acid release and the cleavage of PED6, a synthetic PLA(2) substrate. Such activation can be abolished by p38 MAPK inhibitor. The PEDF-induced PPAR-gamma activation, p53 expression, caspase-3 activity, and apoptosis can be abolished by both cPLA(2) inhibitor and small interfering RNA targeting cPLA(2)-alpha. Our observation not only establishes the signaling role of cPLA(2)-alpha but also for the first time demonstrates the sequential activation of p38 MAPK, cPLA(2)-alpha, PPAR-gamma, and p53 as the mechanism of PEDF-induced endothelial cell apoptosis.
Asunto(s)
Apoptosis , Células Endoteliales/enzimología , Células Endoteliales/patología , Proteínas del Ojo/metabolismo , Fosfolipasas A2 Grupo IV/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Serpinas/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular , Animales , Apoptosis/efectos de los fármacos , Ácido Araquidónico/metabolismo , Quemaduras Químicas/enzimología , Quemaduras Químicas/patología , Caspasa 3/metabolismo , Células Cultivadas , Neovascularización de la Córnea/enzimología , Neovascularización de la Córnea/patología , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Quemaduras Oculares/inducido químicamente , Quemaduras Oculares/enzimología , Quemaduras Oculares/patología , Proteínas del Ojo/administración & dosificación , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Fosfolipasas A2 Grupo IV/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Factores de Crecimiento Nervioso/administración & dosificación , PPAR gamma/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Proteínas Recombinantes/metabolismo , Serina , Serpinas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
PURPOSE: Prostaglandin E2 is related to wound healing. Three different prostaglandin E synthases have been identified: microsomal prostaglandin E synthase (mPGES)-1, mPGES-2, and cytosolic prostaglandin E synthase. This study examined mPGES-1 expression in the cornea during the reparative process that occurs after an alkali burn. METHODS: mPGES-1 messenger RNA (mRNA) and protein expression levels were examined by reverse transcription-polymerase chain reaction and Western blot analysis. Localization of mPGES-1 mRNA was examined by in situ hybridization. Using immunostaining, the localization of mPGES-1, cyclooxygenase (COX)-2, and alpha-smooth muscle actin (alpha-SMA) protein was studied. RESULTS: Although mPGES-1 mRNA is expressed in normal cornea, after a corneal injury, a progressive increase of mPGES-1 mRNA occurs. In this study, 2-6 weeks after injury, mPGES-1 mRNA was detected in the stromal spindle cells. Western blot analysis also showed that mPGES-1 protein expression was observed in normal cornea, with an increase noted from 2 to 4 weeks after corneal injury. mPGES-1 immunoreactivity was negative in normal cornea; however, starting at 2 weeks after injury, positive staining of the stromal spindle cells was noted. Although COX-2 and alpha-SMA immunoreactivities were negative in the stroma of normal cornea, after injury, staining was observed in the stromal spindle cells. CONCLUSIONS: alpha-SMA-positive cells and myofibroblasts express mPGES-1 mRNA and protein, and in addition, mPGES-1 colocalized with COX-2, suggesting that myofibroblasts synthesize prostaglandin E2 and may act on and accelerate corneal wound healing.
Asunto(s)
Álcalis , Quemaduras Químicas/enzimología , Lesiones de la Cornea , Quemaduras Oculares/enzimología , Fibroblastos/enzimología , Oxidorreductasas Intramoleculares/metabolismo , Actinas/metabolismo , Animales , Quemaduras Químicas/metabolismo , Córnea/enzimología , Córnea/metabolismo , Ciclooxigenasa 2/metabolismo , Quemaduras Oculares/inducido químicamente , Quemaduras Oculares/metabolismo , Oxidorreductasas Intramoleculares/genética , Masculino , Músculo Liso/metabolismo , Prostaglandina-E Sintasas , ARN Mensajero/metabolismo , Conejos , Distribución TisularRESUMEN
The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 microg protein, gels were incubated with 50, 100, or 250 microg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09%). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50% after incubation with 50 microg/mL and was completely abolished at 100 and 250 microg/mL of the extract. After incubation with 50 microg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50%. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 microg/mL of the extract. For MMP-2 the incubation with 100 or 250 microg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.
Asunto(s)
Quemaduras Químicas/enzimología , Lesiones de la Cornea , Inhibidores Enzimáticos/farmacología , Quemaduras Oculares/inducido químicamente , Inhibidores de la Metaloproteinasa de la Matriz , Piperaceae/química , Animales , Córnea/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/aislamiento & purificación , Quemaduras Oculares/enzimología , Metaloproteinasas de la Matriz/metabolismo , Fitoterapia , Extractos Vegetales/farmacología , ConejosRESUMEN
The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 æg protein, gels were incubated with 50, 100, or 250 µg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09 percent). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50 percent after incubation with 50 µg/mL and was completely abolished at 100 and 250 µg/mL of the extract. After incubation with 50 µg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50 percent. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 µg/mL of the extract. For MMP-2 the incubation with 100 or 250 µg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.
Asunto(s)
Animales , Conejos , Quemaduras Químicas/enzimología , Córnea/lesiones , Inhibidores Enzimáticos/farmacología , Quemaduras Oculares/inducido químicamente , Metaloproteinasas de la Matriz/antagonistas & inhibidores , Piperaceae/química , Córnea/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/aislamiento & purificación , Quemaduras Oculares/enzimología , Metaloproteinasas de la Matriz/metabolismo , Fitoterapia , Extractos Vegetales/farmacologíaRESUMEN
Corneal wound healing often leads to the development of scar tissue with loss of transparency. Reconstitution of transparent corneal stroma depends on the regulation of the biosynthetic activities of postlesional keratocytes and also to a large extent on the limitation of matrix degradation, attributed essentially to the upregulation of matrix metalloproteases and especially MMP-9. Using a standardized method for the production of reproducible corneal lesions by burning with iodine vapors, we could show that the local application of 0.5 mg/ml L-fucose reduced significantly MMP-9 upregulation and accelerated the recovery of the epithelial layer of the cornea. The iodine vapor used in the experiments produces a rapid loss of epithelium with no or slight effect below the basement membrane. A relatively rapid regrowth of epithelium was observed. The speed of this reepithelialization was stimulated by the local application of fucose. At 48 h after burn, there was a difference between fucose-treated and control corneas (epithelial thickness was about 50 mum for fucose-treated corneas and 37 microm for control corneas). Culture media of in vivo fucose-treated corneas showed an important decrease of MMP-9 activity (-51%, n = 6, p < 0.01). It appears that the in vivo fucose treatment reduced the MMP-9 activity released in the media. This effect is significant 24 h after iodine vapor burn. In order to study the effect of fucose on normal corneas, it was added to rabbit as well as human cornea explant cultures, and the production and release of MMP-9 was determined by zymography. Fucose at a concentration of 0.5 mg/ml produced a 70% decrease of MMP-9 activity released in the medium by corneal explant cultures. Other mono- and oligosaccharides were also tested. Besides lactose, fucose-rich oligosaccharides also produced significant inhibition. Galactose, melibiose, mannose and glucose were inactive. These results justify the use of fucose for the local treatment of corneal wounds.
Asunto(s)
Quemaduras Químicas/tratamiento farmacológico , Epitelio Corneal/efectos de los fármacos , Quemaduras Oculares/inducido químicamente , Fucosa/uso terapéutico , Metaloproteinasa 9 de la Matriz/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Animales , Quemaduras Químicas/enzimología , Quemaduras Químicas/patología , Regulación hacia Abajo , Epitelio Corneal/ultraestructura , Quemaduras Oculares/tratamiento farmacológico , Quemaduras Oculares/enzimología , Yodo/toxicidad , Técnicas de Cultivo de Órganos , ConejosRESUMEN
PURPOSE: Human amniotic membranes have recently been used in ophthalmology to restore deleted ocular surface after burns. Matrix metalloproteinases-2 and -9 have been implicated in the development of neovascularization. In this study, MMP-2 and MMP-9 expression was analyzed by in situ zymography on rabbit corneal chemical burns with and without human amniotic membrane graft. METHOD: Corneal neovascularization was induced in 10 Fauve de Bourgogne rabbits by means of a heptanol chemical burn on controlled deep keratotomy using a Chiron ALK-E corneal shaper. Half of rabbits received acute amniotic membrane transplantation 30mn after chemical burn; the remaining five rabbits received medical treatment. In situ zymography is a recent nondestructive technique which preserved the fine morphological details of the cornea and showed the active enzyme location in different corneal layers. The MMP-2 and -9 substrate was gelatin, which was detected by fluorescent microscopy. RESULTS: There was an overexpression of MMP-2 and -9 in corneal burns versus control corneas. Expression of MMP-2 and -9 was low in corneal burn without amniotic membrane graft. Following amniotic membrane transplantation, MMP-2 and -9 were strongly expressed and clinical neovascularization and inflammation decreased. Active enzymes were located in epithelium layers in the uncovered group. In the covered group, the active enzymes were located in the anterior and posterior stromal layers. CONCLUSION: The results support a role for MMP-2 and MMP-9 in corneal burn neovascularization. Amniotic membrane transplantation can play a protective role by up-regulation of their biological expression.
Asunto(s)
Amnios/trasplante , Córnea/irrigación sanguínea , Quemaduras Oculares/enzimología , Quemaduras Oculares/cirugía , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Animales , Quemaduras Químicas , Colagenasas/metabolismo , Modelos Animales de Enfermedad , Gelatinasas/metabolismo , Humanos , Neovascularización Fisiológica , Conejos , Trasplante HeterólogoRESUMEN
PURPOSE: To demonstrate the potential value of tetracyclines in the treatment of corneal ulceration after moderate to severe ocular chemical injuries. METHODS: Review of published materials describing landmarks in the development of tetracyclines as matrix metalloproteinase inhibitors in ophthalmology and related disciplines. RESULTS: Tetracyclines can protect the cornea against proteolytic degradation after moderate to severe ocular chemical injury. They inhibit matrix metalloproteinases by mechanisms independent of their antimicrobial properties, primarily through restriction of the gene expression of neutrophil collagenase and epithelial gelatinase, suppression of alpha1-antitrypsin degradation, and scavenging of reactive oxygen species. CONCLUSION: Oral tetracyclines can be used along with topical tetracycline preparations and other therapeutic agents to inhibit collagenolytic degradation of the cornea after moderate to severe ocular chemical injuries.
Asunto(s)
Quemaduras Químicas/tratamiento farmacológico , Sustancia Propia/efectos de los fármacos , Úlcera de la Córnea/tratamiento farmacológico , Quemaduras Oculares/inducido químicamente , Tetraciclinas/uso terapéutico , Animales , Quemaduras Químicas/enzimología , Úlcera de la Córnea/inducido químicamente , Úlcera de la Córnea/enzimología , Quemaduras Oculares/enzimología , Fibroblastos/enzimología , Depuradores de Radicales Libres , Humanos , Metaloendopeptidasas/antagonistas & inhibidores , Neutrófilos/enzimología , Cicatrización de HeridasRESUMEN
Amniotic membrane (AM) contains basement membrane components and various proteinase inhibitors. Furthermore, when used as a graft, the basement membrane of AM could block inflammatory insults to a damaged corneal surface. Thus, we evaluated whether amniotic membrane patching could promote the healing process by inhibiting proteolytic damage. Alkali wounds were inflicted on the central corneas of rabbits by applying a round filter paper, 6.0 mm in diameter, soaked in 1 N NaOH for 30 sec. Amniotic membrane patching was performed over the perilimbal sclera immediately after wounding. A total of 115 rabbits were divided into four groups: (1) immediately covered by AM with the amnion cell side down up to the perilimbal sclera (n =26); (2) covered by AM with the stromal side down up to the perilimbal sclera (n =19); (3) anchored to the fornix (n =29); and (4) uncovered as a control (n =41). AM was removed 3 days postoperatively. During follow-ups, epithelial defects, corneal thickness and its opacity of each eye were measured. Some corneas were removed for histopathologic studies and for proteinase activity assay and zymography. The epithelial healing was faster and the corneal thickness was thicker in all three AM-covered groups than in the control (P<0.05). No significant difference was found between covered and anchored groups (P>0.05). Corneal opacity was least in the amnion cell side down group. Infiltration of polymorphonuclear leukocytes (PMNs) was much less in AM-covered groups than in the control. Pathological results were associated with zymographic findings, which revealed much higher proteinase activity in uncovered group than AM-covered groups. Immediate intervention for acute alkali burns with AM as a temporary patch promotes wound healing by inhibiting proteinase activity and PMNs infiltration.
Asunto(s)
Apósitos Biológicos , Quemaduras Químicas/terapia , Lesiones de la Cornea , Endopeptidasas/metabolismo , Quemaduras Oculares/terapia , Animales , Quemaduras Químicas/enzimología , Quemaduras Químicas/patología , Córnea/enzimología , Córnea/patología , Opacidad de la Córnea/terapia , Quemaduras Oculares/enzimología , Quemaduras Oculares/patología , Femenino , Masculino , Conejos , Cicatrización de HeridasRESUMEN
PURPOSE: Inducible nitric oxide synthase (NOS-II) is expressed in many inflammatory conditions. The implication of nitric oxide (NO) in angiogenesis remains controversial. The role of NOS-II and its influence on angiogenesis in corneal neovascularization is unknown and was investigated in this study. METHODS: A mouse model of corneal neovascularization induced by chemical cauterization was used. NOS-II mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction, and NOS-II protein was studied in situ by immunohistochemical analysis of the cornea. The influence of NOS-II on neovascularization was determined by comparison of vessel development in "normal" wild-type mice and mice with a targeted disruption of the NOS-II gene. RESULTS: NOS-II mRNA was induced to very high levels after corneal cauterization and remained upregulated throughout the disease. Migratory cells in the center of the cauterization area expressed NOS-II protein. The neovascular response in mice lacking the NOS-II gene was significantly stronger than in wild-type mice, and the difference increased over time. CONCLUSIONS: These data are the first evidence that NOS-II is expressed in this model of sterile corneal inflammation. NOS-II expression inhibited angiogenesis in severe corneal alkali burns.
Asunto(s)
Quemaduras Químicas/enzimología , Córnea/enzimología , Neovascularización de la Córnea/prevención & control , Quemaduras Oculares/inducido químicamente , Óxido Nítrico Sintasa/biosíntesis , Animales , Quemaduras Químicas/patología , Córnea/efectos de los fármacos , Córnea/patología , Neovascularización de la Córnea/inducido químicamente , Neovascularización de la Córnea/enzimología , Neovascularización de la Córnea/patología , Cartilla de ADN/química , Quemaduras Oculares/enzimología , Quemaduras Oculares/patología , Femenino , Expresión Génica , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nitrato de PlataRESUMEN
PURPOSE: To assess whether changes in lactate dehydrogenase (LDH) activity, as a marker of cell damage, could be detected in corneal stroma and corneal epithelium after toxicity measurements using the enucleated eye test (EET). METHODS: The corneal surface of isolated bovine eyes was continuously wetted with commercial balanced salt solution (BSS) over 4 h at 37 degrees C and central corneal thickness (CCT) was measured. Initial 5-min challenges were made with dilute solutions of benzalkonium chloride (up to 0.032%) or NaOH (up to 0.4 M). The residual LDH activity in the corneal epithelium and corneal stroma were then assessed. RESULTS: Dose-dependent increases in CCT of up to 70% were measured. A strong correlation (r = -0.961) was found between increases in CCT and decreases in stromal LDH activity, but the correlation was much less (r = -0.512) for epithelial LDH activity. CONCLUSIONS: LDH activity can be used as one marker for cell integrity in the corneal stroma and epithelium, although dose-dependent correlations may not be high.