RESUMEN
Purpose: Tissue plasminogen activator (tPA) prevents steroid-induced reduction in aqueous humor outflow facility; however, its mechanism of action at the trabecular meshwork (TM) remains unclear. Enzymatic and non-enzymatic domains allow tPA to function as both an enzyme and a cytokine. This study sought to determine whether cytokine activity is sufficient to rescue steroid-induced outflow facility reduction. Methods: Outflow facility was measured in C57BL/6J mice following triamcinolone acetonide exposure and either transfection of the TM using adenoviral vectors, encoding for enzymatically active and inactive tPA, or administration of the respective proteins. Protein injections were also administered to tPA deficient (PlatKO) and Mmp-9 deficient (Mmp-9KO) mice to determine the potential to rescue reductions in outflow facility and determine downstream mechanisms. Gene expression of matrix metalloproteinases (Mmp-2, -9, and -13) was measured in angle ring tissues containing the TM. Results: Enzymatically active and inactive tPA (either produced after TM transfection or after direct administration) were equally effective in attenuating steroid-induced outflow facility reduction in C57BL/6J mice. They were also equally effective in rescuing outflow reduction in PlatKO mice and causing enhanced expression of matrix metalloproteinases. However, both enzymatically active and enzymatically inactive tPA did not improve outflow reduction in Mmp-9KO mice or increase the baseline outflow facility in naïve C57BL/6J mice. Conclusions: tPA enzymatic activity is not necessary in the regulation of aqueous humor outflow. tPA can increase the expression of matrix metalloproteinases in a cytokine-mediated fashion. This cascade of events may eventually lead to extracellular matrix remodeling at the TM, which reverses outflow facility reduction caused by steroids.
Asunto(s)
Presión Intraocular , Activador de Tejido Plasminógeno , Animales , Humor Acuoso , Ratones , Ratones Endogámicos C57BL , Esteroides , Malla TrabecularRESUMEN
Tissue plasminogen activator (tPA) has been shown to prevent steroid-induced reduction in aqueous humor outflow facility via an upregulation in matrix metalloproteinase (Mmp) expression. The purpose of this study was to determine whether tPA can rescue outflow facility reduction in the Tg-MYOCY437H mouse model, which replicates human juvenile open angle glaucoma. Outflow facility was measured in Tg-MYOCY437H mice following: periocular steroid exposure and intraocular protein treatment with enzymatically active or enzymatically inactive tPA. Effects of tPA on outflow facility were compared to those of animals treated with topical sodium phenylbutarate (PBA), a modulator of endoplasmic reticulum stress. Gene expression of fibrinolytic pathway components (Plat, Plau, and Pai-1) and matrix metalloproteinases (Mmp-2, -9, and -13) was determined in angle ring tissues containing the trabecular meshwork. Tg-MYOCY437H mice did not display further outflow facility reduction following steroid exposure. Enzymatically active and enzymatically inactive tPA were equally effective in attenuating outflow facility reduction in Tg-MYOCY437H mice and caused enhanced expression of matrix metalloproteinases (Mmp-9 and Mmp-13). tPA was equally effective to topical PBA treatment in ameliorating outflow facility reduction in Tg-MYOCY437H mice. Both treatments were associated with an upregulation in Mmp-9 expression while tPA also upregulated Mmp-13 expression. tPA increases the expression of matrix metalloproteinases and may cause extracellular matrix remodeling at the trabecular meshwork, which results in reversal of outflow facility reduction in Tg-MYOCY437H mice.
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Humor Acuoso/metabolismo , Glaucoma de Ángulo Abierto/tratamiento farmacológico , Presión Intraocular/fisiología , Metaloproteinasas de la Matriz/genética , Activadores Plasminogénicos/farmacología , Malla Trabecular/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Femenino , Glaucoma de Ángulo Abierto/genética , Glaucoma de Ángulo Abierto/fisiopatología , Presión Intraocular/efectos de los fármacos , Masculino , Metaloproteinasas de la Matriz/biosíntesis , Ratones , Ratones Noqueados , Malla Trabecular/metabolismoRESUMEN
There is evidence that genetic polymorphisms and environmentally induced epigenetic changes play an important role in modifying disease risk. The commensal microbiota has the ability to affect the cellular environment throughout the body without requiring direct contact; for example, through the generation of a pro-inflammatory state. In this review, we discuss evidence that dysbiosis in intestinal, pharyngeal, oral, and ocular microbiome can lead to epigenetic reprogramming and inflammation making the host more susceptible to ocular disease such as autoimmune uveitis, age-related macular degeneration, and open angle glaucoma. Several mechanisms of action have been proposed to explain how changes to commensal microbiota contribute to these diseases. This is an evolving field that has potentially significant implications in the management of these conditions especially from a public health perspective.
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Disbiosis/complicaciones , Epigénesis Genética , Oftalmopatías/patología , Microbiota , Animales , Oftalmopatías/etiología , HumanosRESUMEN
Purpose: To understand the role and further dissect pathways downstream of tissue plasminogen activator (tPA) and the fibrinolytic pathway in modulating outflow facility. Methods: Outflow facility of tissue plasminogen activator (Plat) knockout (KO) mice was determined and compared to that of wild-type (WT) littermates. Gene expression of urokinase plasminogen activator (Plau), plasminogen activator inhibitor (Pai-1), plasminogen (Plg), and matrix metalloproteinases (Mmp-2, -9, and -13) was measured in angle tissues. Expression of the same genes and outflow facility were measured in KO and WT mice treated with triamcinolone acetonide (TA). Amiloride was used to inhibit urokinase plasminogen activator (uPA) in Plat KO mice, and outflow facility was measured. Results: Plat deletion resulted in outflow facility reduction and decreased Mmp-9 expression in angle tissues. Plasminogen expression was undetectable in both KO and WT mice. TA led to further reduction in outflow facility and decreases in expression of Plau and Mmp-13 in plat KO mice. Amiloride inhibition of uPA activity prevented the TA-induced outflow facility reduction in Plat KO mice. Conclusions: tPA deficiency reduced outflow facility in mice and was associated with reduced MMP expression. The mechanism of action of tPA is unlikely to involve plasminogen activation. tPA is not the only mediator of TA-induced outflow facility change, as TA caused reduction in outflow facility of Plat KO mice. uPA did not substitute for tPA in outflow facility regulation but abrogated the effect of TA in the absence of tPA, suggesting a complex role of components of the fibrinolytic system in outflow regulation.
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Fibrinólisis/fisiología , Inhibidor 1 de Activador Plasminogénico/fisiología , Plasminógeno/fisiología , Activador de Tejido Plasminógeno/fisiología , Malla Trabecular/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Amilorida/farmacología , Animales , Diuréticos/farmacología , Regulación de la Expresión Génica/fisiología , Glucocorticoides/farmacología , Inyecciones Intraoculares , Presión Intraocular/fisiología , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Malla Trabecular/efectos de los fármacos , Triamcinolona Acetonida/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidoresRESUMEN
This protocol provides a step-by-step guide to shotgun sphingolipid analysis of aqueous humor. We describe the Bligh and Dyer crude lipid extraction method and electrospray ionization tandem mass spectrometry (ESI-MS/MS) coupled with MZmine 2.21 data processing for identification and ratiometric quantitation of sphingosine, sphingosine-1-phosphate, sphingomyelin, and ceramide.
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Humor Acuoso/química , Esfingolípidos/análisis , Ceramidas/análisis , Humanos , Lisofosfolípidos/análisis , Programas Informáticos , Espectrometría de Masa por Ionización de Electrospray/métodos , Esfingomielinas/análisis , Esfingosina/análogos & derivados , Esfingosina/análisis , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Estrogens exert profound effects on target tissues. These effects are mediated by two estrogen receptors (ER(alpha) and ER(beta)) that bind to specific DNA sequences in estrogen-dependent genes. Other molecules such as growth factors, transcription factors and some oncoproteins might interact with the estrogen receptors and thus regulate the transcription of these genes. Currently there is no adequate cellular model to study these interactions. METHODS: We transfected the human wild-type ER(alpha) to an ER-negative rat epithelial endometrial cell line (Rentr01) using a tetracycline-regulated gene expression system. The exogenous receptor was correctly translated, had an appropriate hormone-binding affinity, and bound well to estrogen response elements containing DNA. RESULTS: We obtained a new stable cell line that is ER(beta) negative but ER(alpha) positive (R1-49E1). The expression of receptor alpha can be regulated in a dose-response manner by addition of tetracycline in the culture medium. Estradiol treatment of ER(alpha)-containing cells apparently diminished cellular proliferation, and the exogenous receptor can induce the transcription of the endogenous progesterone receptor isoform B (PgR-B) gene. CONCLUSIONS: This epithelial cellular model may be useful to study the interaction between estrogens and other cell signaling pathways in epithelial endometrial cell physiology.
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Línea Celular , Endometrio/citología , Receptor alfa de Estrógeno/biosíntesis , Tetraciclina/farmacología , Animales , Diferenciación Celular , Proliferación Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Medios de Cultivo/farmacología , ADN/química , Relación Dosis-Respuesta a Droga , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Femenino , Genes Reporteros , Humanos , Inmunohistoquímica , Ratones , Células 3T3 NIH , Unión Proteica , Ratas , Receptores de Progesterona/metabolismo , Elementos de Respuesta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
INTRODUCCIÓN: El virus del papiloma humano (VPH) es el virus que más frecuentemente se transmite por contacto sexual y está fuertemente asociado con el cáncer cérvicouterino. Los tipos virales que se documentan con mayor frecuencia en el epitelio cervical son 6, 11, 16, 18, 31 y 33, los cuales difieren en su capacidad oncogénica; los dos primeros presentan bajo potencial y los últimos alto potencial oncogénico. Objetivo: Identificar los tipos de VPH presentes en biopsias de cervix con evidencia citológica, colposcópica e histopatológica de infección viral, y realizar un análisis ultraestructural de los cambios celulares asociados. Material y métodos: Usamos la hibridación in situ (HIS) con sondas biotiniladas para identificar los tipos virales, y las biopsias positivas se procesaron para análisis ultraestructural por microscopia electrónica. Resultados: Se analizaron por HIS 63 biopsias cervicales, 24 (38%) resultaron positivas para alguno de los tipos virales probados, los más frecuentes fueron los tipos 6/11 (24%), los cuales en el análisis ultraestructural se asociaron a la presencia de varios nucléolos en núcleos con bordes crenados, en tanto que los tipos oncogénicos se asociaron con la presencia de partículas virales en núcleos, que frecuentemente se presentan multilobulados y con inclusiones citoplasmáticas. En ambos casos es evidente la desorganización de los desmosomas. Conclusiones: La prevalencia de VPH encontrada en este estudio concuerda con lo reportado para esta infección viral en otros estudios donde se usa la misma técnica, los tipos virales concuerdan con el diagnóstico inicial, y el estudio ultraestructural arroja evidencias sobre las principales alteraciones celulares asociadas a la infección viral.
Introduction: Human papillomavirus (HPV) is the most common sexually transmitted virus, is strongly associated with cervical cancer. HPV genital types differs in oncogenic potential, types 6/11 are low-risk and, types 16/18, 31/33 are high-risk. In situ Hybridization (ISH) analysis has advantage of being able to detect both, the specific cellular types infected with viral DNA and the HPV types in cervical biopsies and therefore, cervical cancer patient's risk. Objective: This work was performed in order to typing HPV in cervical biopsies with cytological, colposcopical and histopathological evidence of HPV infection, and to search ultrastructural changes associates with viral infection. Results: We analyzed, by ISH, 63 cervical biopsies, 24 (38%) positives to some viral type, HPV types 6/11 were more common types (24%) consistent with the more frequent, both colposcopical and histopathological diagnostic; Low-grade intraepithelial lesion. Ultraestructural analysis showed cellular alteration in association with low-risk types, principally irregular nucleus and several nucleolus for nucleus, as long as cells infected with oncogenic types DNA showed multilobulated nucleus, cytoplasmic inclusions and viral particles in nucleus. In both cases is clear an disorganization of cellular junctions desmosomes. Conclusions: The HPV prevalence in this study was consistent with other reported, using ISH analysis, the more common types were low-risk types in agreement with previous diagnostic, and electron microscopy analysis provide evidence on the main cell alterations associated with viral infection.
RESUMEN
Objetivo. Estudiar el efecto de 1,25-dihydroxycholecalciferol D(1,25-(OH)2D3 sobre la proliferación y muerte de las células de endometrio de la rata en cultivo. Material y métodos. Se usó la línea celular de endometrio de rata Rentro 1. El medio de incubación se suplementó con 1 por ciento de suero bovino fetal inactivado y previamente tratado con carbón para eliminar las hormonas esteroides. Las monocapas de células fueron mantenidas en presencia o ausencia de 1,25-(OH)2D3 o 17ß-estradiol o del vehículo. Posteriormente se evaluó la proliferación celular mediante conteo en un hemocitómetro, utilizando azul tripano 0.4 por ciento y se analizó la fase de síntesis de ADN por citofluorometría fe flujo. La muerte celular fue determinada por el análisis de la integridad del ADN genómico en geles de agarosa y tinción con bromuro de etidio. Resultados. Las células en presencia de 1 por ciento de suero bovina fetal sin hormonas esteroides en el medio de cultivo, estimuló su crecimiento de las mismas. Por otro lado, las células Rentro 1 no respondieron a la estimulación con 17ß-estradiol y sí al 1,25-(OH)2D3, lo que confirmó la ausencia del receptor de estrógenos en estas células y demostró la capacidad de esta línea celular para responder al 1,25-(OH)2D3. Por último, se encontró que a diferencia de otros tipos celulares, las células Rentro 1 no sufrieron daño a nivel del ADN (apoptosis) con el 1,25-(OH)2D3. Conclusiones. 1) El 1,25-(OH)2D3 promovió la proliferación de las células Rentro 1 de manera independiente de la dosis e independiente de la presencia del estímulo estrogénico; 2) el incremento en el número de células estuvo en relación con la activación de la fase de síntesis de ADN del ciclo celular; 3) la presencia de esta hormona en el cultivo celular no indujo la muerte celular no indujo la muerte celular por apoptosis
Asunto(s)
Animales , Femenino , Ratas , Calcitriol/farmacología , Ciclo Celular , Línea Celular , Células Cultivadas , División Celular , ADN/biosíntesis , Endometrio/citología , Endometrio/efectos de los fármacos , Vitamina D/farmacologíaRESUMEN
Se analizaron once placentas provenientes de mujeres seropositivas para VIH. en tres casos el material provino de abortos del primer trimestre y en los ocho restantes de embarazos a término. En cinco casos se identificaron retrovirus semejantes a VIH en tejido placentario. Se demostró por primera vez la internalización de un retrovirus y su presencia en el sincitiotrofoblasto. Se comunica por primera vez la presencia de una célula en el estroma placentario diferente a la de Hofbauer por su tipo de gránulos