RESUMEN
Purpose: Transgenic mice overexpressing serum retinol-binding protein (RBP4-Tg) develop progressive retinal degeneration, characterized by microglia activation, yet the precise mechanisms underlying retinal degeneration are unclear. Previous studies showed RBP4-Tg mice have normal ocular retinoid levels, suggesting that degeneration is independent of the retinoid visual cycle or light exposure. The present study addresses whether retinal degeneration is light-dependent and RBP4-dependent by testing the effects of dark-rearing and pharmacological lowering of serum RBP4 levels, respectively. Methods: RBP4-Tg mice reared on normal mouse chow in normal cyclic light conditions were directly compared to RBP4-Tg mice exposed to chow supplemented with the RBP4-lowering compound A1120 or dark-rearing conditions. Quantitative retinal histological analysis was conducted to assess retinal degeneration, and electroretinography (ERG) and optokinetic tracking (OKT) tests were performed to assess retinal and visual function. Ocular retinoids and bis-retinoid A2E were quantified. Results: Dark-rearing RBP4-Tg mice effectively reduced ocular bis-retinoid A2E levels, but had no significant effect on retinal degeneration or dysfunction in RBP4-Tg mice, demonstrating that retinal degeneration is light-independent. A1120 treatment lowered serum RBP4 levels similar to wild-type mice, and prevented structural retinal degeneration. However, A1120 treatment did not prevent retinal dysfunction in RBP4-Tg mice. Moreover, RBP4-Tg mice on A1120 diet had significant worsening of OKT response and loss of cone photoreceptors compared to RBP4-Tg mice on normal chow. This may be related to the very significant reduction in retinyl ester levels in the retina of mice on A1120-supplemented diet. Conclusions: Retinal degeneration in RBP4-Tg mice is RBP4-dependent and light-independent.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Traumatismos Experimentales por Radiación/genética , Retina/efectos de la radiación , Degeneración Retiniana/genética , Proteínas Plasmáticas de Unión al Retinol/genética , Animales , Adaptación a la Oscuridad , Electrorretinografía , Femenino , Luz , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Piperidinas/farmacología , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Retinoides/metabolismo , Proteínas Plasmáticas de Unión al Retinol/antagonistas & inhibidores , Proteínas Plasmáticas de Unión al Retinol/metabolismoRESUMEN
Purpose: Diabetic retinopathy is a leading cause of vision loss. Previous studies have shown signaling pathways mediated by Stat3 (signal transducer and activator of transcription 3) play a primary role in diabetic retinopathy progression. This study tested CLT-005, a small molecule inhibitor of Stat3, for its dose-dependent therapeutic effects on vision loss in a rat model of diabetic retinopathy. Methods: Brown Norway rats were administered streptozotocin (STZ) to induce diabetes. CLT-005 was administered daily by oral gavage for 16 weeks at concentrations of 125, 250, or 500 mg/kg, respectively, beginning 4 days post streptozotocin administration. Systemic and ocular drug concentration was quantified with mass spectrometry. Visual function was monitored at 2-week intervals from 6 to 16 weeks using optokinetic tracking to measure visual acuity and contrast sensitivity. The presence and severity of cataracts was visually monitored and correlated to visual acuity. The transcription and translation of multiple angiogenic factors and inflammatory cytokines were measured by real-time polymerase chain reaction and Multiplex immunoassay. Results: Streptozotocin-diabetic rats sustain progressive vision loss over 16 weeks, and this loss in visual function is rescued in a dose-dependent manner by CLT-005. This positive therapeutic effect correlates to the positive effects of CLT-005 on vascular leakage and the presence of inflammatory cytokines in the retina. Conclusions: The present study indicates that Stat3 inhibition has strong therapeutic potential for the treatment of vision loss in diabetic retinopathy.
Asunto(s)
Ceguera/prevención & control , Diabetes Mellitus Experimental , Retinopatía Diabética/tratamiento farmacológico , Proteínas Inhibidoras de STAT Activados/uso terapéutico , Factor de Transcripción STAT3/antagonistas & inhibidores , Agudeza Visual , Animales , Ceguera/etiología , Ceguera/fisiopatología , Retinopatía Diabética/complicaciones , Retinopatía Diabética/fisiopatología , Femenino , Immunoblotting , Ratas , Ratas Endogámicas BNRESUMEN
PURPOSE: To provide a comprehensive study of the biological role and therapeutic potential of six endogenous epidermal growth factor receptor (EGFR) ligands in corneal epithelial homeostasis. METHODS: Kinetic analysis and dose response curves were performed by using in vitro and in vivo wound-healing assays. Biochemical assays were used to determine receptor expression and activity. Human tears were collected and quantitatively analyzed by multianalyte profiling for endogenous EGFR ligands. RESULTS: Epidermal growth factor receptor ligands improved wound closure and activated EGFR, but betacellulin (BTC) was the most efficacious promoter of wound healing in vitro. In contrast, only epidermal growth factor (EGF) promoted wound healing in vivo. Human tears from 25 healthy individuals showed EGFR ligands at these average concentrations: EGF at 2053 ± 312.4 pg/mL, BTC at 207 ± 39.4 pg/mL, heparin-binding EGF at 44 ± 5.8 pg/mL, amphiregulin at 509 ± 28.8 pg/mL, transforming growth factor-α at 84 ± 19 pg/mL, and epiregulin at 52 ± 15 pg/mL. CONCLUSIONS: Under unwounded conditions, only EGF was present at concentrations near the ligand's Kd for the receptor, indicating it is the primary mediator of corneal epithelial homeostasis. Other ligands were present but at concentrations 11- to 7500-fold less their Kd, preventing significant ligand binding. Further, the high levels of EGF and its predicted binding preclude receptor occupancy by exogenous ligand and can explain the discrepancy between the in vitro and in vivo data. Therefore, therapeutic use of EGFR ligands may be unpredictable and impractical.
Asunto(s)
Epitelio Corneal/fisiología , Receptores ErbB/fisiología , Homeostasis/fisiología , Adulto , Animales , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Modelos Biológicos , Lágrimas/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Cicatrización de Heridas/fisiología , Adulto JovenRESUMEN
Toxoplasma gondii is a wide spread pathogen that can cause severe and even fatal disease in fetuses and immune-compromised hosts. As an obligate intracellular parasite, Toxoplasma must alter the environment of its host cell in order to establish its replicative niche. This is accomplished, in part, by secretion of factors into the host cell that act to modulate processes such as transcription. Previous studies demonstrated that genes encoding transcription factors such as c-jun, junB, EGR1, and EGR2 were amongst the host genes that were the most rapidly upregulated following infection. In cells stimulated with growth factors, these genes are regulated by a transcription factor named Serum Response Factor. Serum Response Factor is a ubiquitously expressed DNA binding protein that regulates growth and actin cytoskeleton genes via MAP kinase or actin cytoskeletal signaling, respectively. Here, we report that Toxoplasma infection leads to the rapid activation of Serum Response Factor. Serum Response Factor activation is a Toxoplasma-specific event since the transcription factor is not activated by the closely related protozoan parasite, Neospora caninum. We further demonstrate that Serum Response Factor activation requires a parasite-derived secreted factor that signals via host MAP kinases but independently of the host actin cytoskeleton. Together, these data define Serum Response Factor as a host cell transcription factor that regulates immediate early gene expression in Toxoplasma-infected cells.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Regulación de la Expresión Génica , Interacciones Huésped-Parásitos/genética , Factor de Respuesta Sérica/metabolismo , Toxoplasma/fisiología , Toxoplasmosis/parasitología , Animales , Línea Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Parásitos/fisiología , Proteínas Protozoarias/metabolismo , Transducción de Señal/genética , Factores de Transcripción TCF/metabolismo , Toxoplasmosis/genéticaRESUMEN
Toxoplasma gondii is a ubiquitous apicomplexan parasite that can cause severe disease in fetuses and immune-compromised patients. Rhoptries, micronemes, and dense granules, which are secretory organelles unique to Toxoplasma and other apicomplexan parasites, play critical roles in parasite growth and virulence. To understand how these organelles modulate infected host cells, we sought to identify host cell transcription factors triggered by their release. Early growth response 2 (EGR2) is a host cell transcription factor that is rapidly upregulated and activated in Toxoplasma-infected cells but not in cells infected with the closely related apicomplexan parasite Neospora caninum. EGR2 upregulation occurred only when live parasites were in direct contact with the host cell and not from exposure to cell extracts that contain dense granule or micronemal proteins. When microneme-mediated attachment was blocked by pretreating parasites with a calcium chelator, EGR2 expression was significantly reduced. In contrast, when host cells were infected with parasites in the presence of cytochalasin D, which allows rhoptry secretion but prevents parasite invasion, EGR2 was activated. Finally, we demonstrate that Toxoplasma activation of host p38 mitogen-activated protein kinase is necessary but not sufficient for EGR2 activation. Collectively, these data indicate that EGR2 is specifically upregulated by a parasite-derived secreted factor that is most likely a resident rhoptry protein.
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Proteína 2 de la Respuesta de Crecimiento Precoz/biosíntesis , Orgánulos/fisiología , Proteínas Protozoarias/metabolismo , Toxoplasma/fisiología , Animales , Línea Celular , Humanos , Ratones , Neospora/fisiología , Toxoplasma/citología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Angiopoietins play a significant role in vascular development and angiogenesis. Both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) bind the receptor tyrosine kinase Tie2. However, while Ang1 signaling results in the stabilization of vessel structure, Ang2 has been linked to vascular instability. The ratio of these two Tie2 ligands is thus critical for vascular stability and remodeling. This study identifies a mechanism of growth factor-mediated reduction in Ang2 expression in vascular smooth muscle cells (VSMCs). In response to PDGF, VSMCs downregulated Ang2 mRNA levels by 75% within 4 h, with a subsequent decrease in Ang2 protein levels. Quantitation of endogenous transcription rates revealed that PDGF stimulation did not alter Ang2 transcription rates, but instead induced a posttranscriptional mechanism of rapid Ang2 mRNA destabilization. The Ang2 mRNA half-life was reduced by at least 50% after PDGF treatment. The PDGF-induced mRNA turnover mechanism was dependent on several MAPK pathways, including ERK and JNK. In contrast, IGF-I, which did not significantly activate ERK or JNK, stimulated increased Ang2 expression through transcriptional activation. These findings demonstrate that VSMCs adjust Ang2 expression through multiple mechanisms, including changes in transcription as well as posttranscriptional mRNA destabilization.
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Angiopoyetina 2/metabolismo , Regulación de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Angiopoyetina 2/genética , Animales , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Miocitos del Músculo Liso/citología , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas WKY , Transducción de Señal/fisiologíaRESUMEN
Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process. In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3. Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.