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Introduction: Cerebrovascular pathologies contribute to cognitive decline during aging, leading to vascular cognitive impairment and dementia (VCID). Levels of circulating insulin-like growth factor 1 (IGF-1), a vasoprotective hormone, decrease during aging. Decreased circulating IGF-1 in animal models leads to the development of VCID-like symptoms, but the cellular mechanisms underlying IGF-1-deficiency associated pathologies in the aged cerebrovasculature remain poorly understood. Here, we test the hypothesis that vascular smooth muscle cells (VSMCs) play an integral part in mediating the vasoprotective effects of IGF-1. Methods: We used a hypertension-based model of cerebrovascular dysfunction in mice with VSMC-specific IGF-1 receptor (Igf1r) deficiency and evaluated the development of cerebrovascular pathologies and cognitive dysfunction. Results: VSMC-specific Igf1r deficiency led to impaired cerebral myogenic autoregulation, independent of blood pressure changes, which was also associated with impaired spatial learning and memory function as measured by radial arm water maze and impaired motor learning measured by rotarod. In contrast, VSMC-specific IGF-1 receptor knockdown did not lead to cerebral microvascular rarefaction. Discussion: These studies suggest that VSMCs are key targets for IGF-1 in the context of cerebrovascular health, playing a role in vessel stability alongside other cells in the neurovascular unit, and that VSMC dysfunction in aging likely contributes to VCID.
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OBJECTIVE: Vascular smooth muscle cell (VSMC) phenotypic switching is critical for normal vessel formation, vascular stability, and healthy brain aging. Phenotypic switching is regulated by mediators including platelet derived growth factor (PDGF)-BB, insulin-like growth factor (IGF-1), as well as transforming growth factor-ß (TGF-ß) and endothelin-1 (ET-1), but much about the role of these factors in microvascular VSMCs remains unclear. METHODS: We used primary rat microvascular VSMCs to explore PDGF-BB- and IGF-1-induced phenotypic switching. RESULTS: PDGF-BB induced an early proliferative response, followed by formation of polarized leader cells and rapid, directionally coordinated migration. In contrast, IGF-1 induced cell hypertrophy, and only a small degree of migration by unpolarized cells. TGF-ß and ET-1 selectively inhibit PDGF-BB-induced VSMC migration primarily by repressing migratory polarization and formation of leader cells. Contractile genes were downregulated by both growth factors, while other genes were differentially regulated by PDGF-BB and IGF-1. CONCLUSIONS: These studies indicate that PDGF-BB and IGF-1 stimulate different types of microvascular VSMC phenotypic switching characterized by different modes of cell migration. Our studies are consistent with a chronic vasoprotective role for IGF-1 in VSMCs in the microvasculature while PDGF is more involved in VSMC proliferation and migration in response to acute activities such as neovascularization. Better understanding of the nuances of the phenotypic switching induced by these growth factors is important for our understanding of a variety of microvascular diseases.
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Factor I del Crecimiento Similar a la Insulina , Ratas , Animales , Becaplermina/farmacología , Proteínas Proto-Oncogénicas c-sis/farmacología , Proteínas Proto-Oncogénicas c-sis/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Miocitos del Músculo Liso , Proliferación Celular , Movimiento Celular , Células CultivadasRESUMEN
As a result of their unique compositions and properties, nanomaterials have recently seen a tremendous increase in use for novel cancer therapies. By taking advantage of the optical absorption of near-infrared light, researchers have utilized nanostructures such as carbon nanotubes, gold nanorods, and graphene oxide sheets to enhance photothermal therapies and target the effect on the tumor tissue. However, new uses for nanomaterials in targeted cancer therapy are coming to light, and the efficacy of photothermal therapy has increased dramatically. In this work, we review some of the current applications of nanomaterials to enhance photothermal therapy, specifically as photothermal absorbers, drug delivery vehicles, photoimmunological agents, and theranostic tools.
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The development of therapeutic methods that can effectively delay tumor growth, inhibit tumor metastases, and protect the host from tumor recurrence still faces challenges. Nanoparticle-based combination therapy may provide an effective therapeutic strategy. Herein, we show that bovine serum albumin (BSA)-bioinspired gold nanorods (GNRs) were loaded with an immunoadjuvant for combined photothermal therapy (PTT) and immunotherapy for the treatment of melanoma. In this work, cetyltrimethylammonium bromide (CTAB)-coated GNRs were successively decorated with polyethylene glycol (PEG) and BSA, and loaded with an immunoadjuvant imiquimod (R837). The synthesized mPEG-GNRs@BSA/R837 nanocomplexes under near-infrared (NIR) irradiation could effectively kill tumors and trigger strong immune responses in treating metastatic melanoma in mice. Furthermore, the nanocomplex-based PTT prevented lung metastasis and induced a strong long-term antitumor immunity to protect the treated mice from tumor recurrence. The nanocomplex-based PTT in combination with immunotherapy may be potentially employed as an effective strategy for the treatment of melanoma and other metastatic cancers.
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Adyuvantes Inmunológicos/uso terapéutico , Oro/química , Melanoma Experimental/terapia , Nanotubos/química , Albúmina Sérica Bovina/química , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Animales , Bovinos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cetrimonio/química , Terapia Combinada , Citocinas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Imiquimod/química , Imiquimod/farmacología , Imiquimod/uso terapéutico , Inmunoterapia , Rayos Infrarrojos , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Fototerapia , Polietilenglicoles/químicaRESUMEN
BACKGROUND AND OBJECTIVES: The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs) are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium. METHODOLOGY/PRINCIPAL FINDINGS: Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2), membrane type 1-MMP (MT1-MMP) and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohistochemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime. CONCLUSIONS/SIGNIFICANCE: MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and activation, tight junction protein cleavage, and subsequent surface cell desquamation and renewal may be orchestrated by nocturnal circadian signals.
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Ritmo Circadiano , Epitelio Corneal/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Uniones Estrechas/metabolismo , Xenopus laevis/metabolismo , Animales , Claudina-4/metabolismo , Epitelio Corneal/patología , Inmunohistoquímica , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Microscopía Confocal , Ocludina/metabolismo , Uniones Estrechas/patología , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
BACKGROUND: The discovery of TCF7L2 as a global type 2 diabetes (T2D) gene has sparked investigations to explore the clinical utility of its variants for guiding the development of new diagnostic and therapeutic strategies. However, interpreting the resulting associations into function still remains unclear. Canonical Wnt signaling regulates ß-catenin and its binding with TCF7L2, which in turn is critical for the production of glucagon-like peptide-1 (GLP-1). This study examines the role of a novel frame-shift insertion discovered in a conserved region of WNT16a, and it is proposed that this mutation affects T2D susceptibility in conjunction with gene variants in TCF7L2. RESULTS: Our results predicted that the insertion would convert the upstream open reading frame in the Wnt16a mRNA to an alternative, in-frame translation initiation site, resulting in the prevention of nonsense-mediated decay, leading to a consequent stabilization of the mutated WNT16a message. To examine the role of Wnt16a in the Wnt signaling pathway, DNA and serum samples from 2,034 individuals (48% with T2D) from the Sikh Diabetes Study were used in this investigation. Prevalence of Wnt16a insertion did not differ among T2D cases (33%) and controls (32%). However, there was a 3.2 fold increase in Wnt16a mRNA levels in pancreatic tissues from the insertion carriers and a significant increase (70%, p < 0.0001) in luciferase activity in the constructs carrying the insertion. The expression of TCF7L2 mRNA in pancreas was also elevated (~23-fold) among the insertion carriers (p=0.003). CONCLUSIONS: Our results suggest synergistic effects of WNT16a insertion and the at-risk 'T' allele of TCF7L2 (rs7903146) for elevating the expression of TCF7L2 in human pancreas which may affect the regulation of downstream target genes involved in the development of T2D through Wnt/ß-catenin/TCF7L2 signaling pathway. However, further studies would be needed to mechanistically link the two definitively.
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Mutación del Sistema de Lectura , Tamización de Portadores Genéticos , Páncreas/metabolismo , Proteína 2 Similar al Factor de Transcripción 7/genética , Proteínas Wnt/genética , Adulto , Anciano , Secuencia de Bases , Estudios de Casos y Controles , Cartilla de ADN , Diabetes Mellitus Tipo 2/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
Cell migration is fundamental to many biological processes, including development, normal tissue remodeling, wound healing, and many pathologies. However, cell migration is a complex process, and understanding its regulation in health and disease requires the ability to manipulate and measure this process quantitatively under controlled conditions. This report describes a simple in vitro assay for quantitative analysis of cell migration in two-dimensional cultures that is an inexpensive alternative to the classic "scratch" assay. The method described utilizes flexible silicone masks fabricated in the lab according to the research demands of the specific experiment to create a cell-free area for cells to invade, followed by quantitative analysis based on widely available microscopic imaging tools. This experimental approach has the important advantage of visualizing cell migration in the absence of the cellular damage and disruption of the substrate that occurs when the "wound" is created in the scratch assay. This approach allows the researcher to study the intrinsic migratory characteristics of cells in the absence of potentially confounding contributions from cellular responses to injury and disruption of cell-substrate interactions. This assay has been used with vascular smooth muscle cells, fibroblasts, and epithelial cell types, but should be applicable to the study of practically any type of cultured cell. Furthermore, this method can be easily adapted for use with fluorescence microscopy, molecular biological, or pharmacological manipulations to explore the molecular mechanisms of cell migration, live cell imaging, fluorescence microscopy, and correlative immunolabeling.
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Movimiento Celular/genética , Fibroblastos/metabolismo , Elastómeros de Silicona/química , Bioensayo , Células Cultivadas , Humanos , Elastómeros de Silicona/metabolismo , Cicatrización de Heridas/genéticaRESUMEN
The contractile phenotype and function of myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized that smooth muscle α-actin expressed in myofibroblasts is critical for its formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin-null and wild-type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a myofibroblast-like distribution and a stress fiber-like pattern, showing that these cells acquired the myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild-type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-ß1. Smooth muscle γ-actin and skeletal muscle α-actin were expressed in smooth muscle α-actin-null myofibroblasts, as shown by immunostaining, real-time polymerase chain reaction, and mass spectrometry. These results show that smooth muscle α-actin is not necessary for myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in myofibroblasts.
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Actinas/metabolismo , Fibroblastos/patología , Adhesiones Focales/patología , Tejido de Granulación/patología , Miofibroblastos/patología , Cicatrización de Heridas , Heridas y Lesiones/patología , Animales , Western Blotting , Diferenciación Celular , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
During wound healing, fibroblasts transition from quiescence to a migratory state, then to a contractile myofibroblast state associated with wound closure. We found that the myofibroblast phenotype, characterized by the expression of high levels of contractile proteins, suppresses the expression of the pro-migratory gene, MMP-2. Fibroblasts cultured in a 3-D collagen lattice and allowed to develop tension showed increased contractile protein expression and decreased MMP-2 levels in comparison to a stress-released lattice. In 2-D cultures, factors that promote fibroblast contractility, including serum or TGF-ß, down-regulated MMP-2. Pharmacologically inducing F-actin disassembly or reduced contractility increased MMP-2 expression, while conditions that promote F-actin assembly suppressed MMP-2 expression. In all cases, changes in MMP-2 levels were inversely related to changes in the contractile marker, smooth muscle α-actin. To determine if the mechanisms involved in contractile protein gene expression play a direct role in MMP-2 regulation, we used RNAi-mediated knock-down of the myocardin-like factors, MRTF-A and MRTF-B, which induced the down-regulation of contractile protein genes by fibroblasts under both serum-containing and serum-free conditions. In the presence of serum or TGF-ß, MRTF-A/B knock-down resulted in the up-regulation of MMP-2; serum-free conditions prevented this increased expression. Together, these results indicate that, while MMP-2 expression is suppressed by F-actin formation, its up-regulation is not simply a consequence of contractile protein down-regulation.
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Fibroblastos/enzimología , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Miofibroblastos/enzimología , Actinas/química , Actinas/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Movimiento Celular/genética , Movimiento Celular/fisiología , Fibroblastos/citología , Fibroblastos/fisiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Factor I del Crecimiento Similar a la Insulina/farmacología , Modelos Biológicos , Miofibroblastos/citología , Miofibroblastos/fisiología , Fenotipo , Multimerización de Proteína , Interferencia de ARN , Ratas , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiologíaRESUMEN
Myofibroblasts are contractile, smooth muscle-like cells that are characterized by the de novo expression of smooth muscle α-actin (SMαA) and normally function to assist in wound closure, but have been implicated in pathological contractures. Transforming growth factor ß-1 (TGF-ß1) helps facilitate the differentiation of fibroblasts into myofibroblasts, but the exact mechanism by which this differentiation occurs, in response to TGF-ß1, remains unclear. Myocardin-related transcription factors A and B (MRTFs, MRTF-A/B) are transcriptional co-activators that regulate the expression of smooth muscle-specific cytoskeletal proteins, including SMαA, in smooth muscle cells and fibroblasts. In this study, we demonstrate that TGF-ß1 mediates myofibroblast differentiation and the expression of a contractile gene program through the actions of the MRTFs. Transient transfection of a constitutively active MRTF-A induced an increase in the expression of SMαA and other smooth muscle-specific cytoskeletal proteins, and an increase in myofibroblast contractility, even in the absence of TGF-ß1. MRTF-A/B knockdown, in TGF-ß1-differentiated myofibroblasts, resulted in decreased smooth muscle-specific cytoskeletal protein expression levels and reduced contractile force generation, as well as a decrease in focal adhesion size and number. These results provide direct evidence that the MRTFs are mediators of myofibroblast differentiation in response to TGF-ß1.
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Diferenciación Celular/genética , Fibroblastos/citología , Miofibroblastos/citología , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Actinas/metabolismo , Animales , Línea Celular , Proteínas del Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Adhesiones Focales/metabolismo , Miofibroblastos/metabolismo , RatasRESUMEN
Toxoplasma gondii is an intracellular protozoan parasite that can cause devastating disease in fetuses and immune-compromised individuals. We previously reported that the alpha subunit of the host cell transcription factor, hypoxia-inducible factor-1 (HIF-1), is up-regulated by infection and necessary for Toxoplasma growth. Under basal conditions, HIF-1alpha is constitutively expressed but rapidly targeted for proteasomal degradation after two proline residues are hydroxylated by a family of prolyl hydroxylases (PHDs). The PHDs are alpha-ketoglutarate-dependent dioxygenases that have low K(m) values for oxygen, making them important cellular oxygen sensors. Thus, when oxygen levels decrease, HIF-1alpha is not hydroxylated, and HIF-1 is activated. How Toxoplasma activates HIF-1 under normoxic conditions remains unknown. Here, we report that Toxoplasma infection increases HIF-1alpha stability by preventing HIF-1alpha prolyl hydroxylation. Infection significantly decreases PHD2 abundance, which is the key prolyl hydroxylase for regulating HIF-1alpha. The effects of Toxoplasma on HIF-1alpha abundance and prolyl hydroxylase activity require activin-like receptor kinase signaling. Finally, parasite growth is severely diminished when signaling from this family of receptors is inhibited. Together, these data indicate that PHD2 is a key host cell factor for T. gondii growth and represent a novel mechanism by which a microbial pathogen subverts host cell signaling and transcription to establish its replicative niche.
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Receptores de Activinas Tipo I/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Toxoplasma/metabolismo , Toxoplasmosis/mortalidad , Animales , Células HeLa , Humanos , Hidroxilación , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Oxígeno/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad ProteicaRESUMEN
During platelet-derived growth factor (PDGF)-BB-mediated recruitment to neovascular sprouts, vascular smooth muscle cells (VSMCs) dedifferentiate from a contractile to a migratory phenotype. This involves the downregulation of contractile markers such as smooth muscle (SM) alpha-actin and the upregulation of promigration genes such as matrix metalloproteinase (MMP)-2. The regulation of MMP-2 in response to PDGF-BB is complex and involves both stimulatory and inhibitory signaling pathways, resulting in a significant delay in upregulation. Here, we provide evidence that the delay in MMP-2 upregulation may be due to the autocrine expression and activation of transforming growth factor (TGF)-beta, which is known to promote the contractile phenotype in VSMCs. Whereas PDGF-BB could induce the loss of stress fibers and focal adhesions, TGF-beta was able to block or reverse this transition to a noncontractile state. TGF-beta did not, however, suppress early signaling events stimulated by PDGF-BB. Over time, though PDGF-BB induced increased TGF-beta1 levels, it suppressed TGF-beta2 and TGF-beta3 expression, leading to a net decrease in the total TGF-beta pool, resulting in the upregulation of MMP-2. Together, these findings indicate that MMP-2 expression is suppressed by a threshold level of active TGF-beta, which in turn promotes a contractile VSMC phenotype that prevents the upregulation of MMP-2.
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Metaloproteinasa 2 de la Matriz/genética , Músculo Liso Vascular/enzimología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Factor de Crecimiento Transformador beta1/farmacología , Regulación hacia Arriba/efectos de los fármacos , Actinas/genética , Animales , Becaplermina , Cartilla de ADN , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes fos , Humanos , Factor Estimulante de Colonias de Macrófagos/farmacología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-sis , Ratas , Ratas Endogámicas WKY , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-2/genéticaRESUMEN
PURPOSE: Cellular retinaldehyde-binding protein (CRALBP), transcribed from the RLBP1 gene, is a 36-kDa water-soluble protein with 316 amino acids found in the retinal pigment epithelium (RPE) and in retinal Müller cells. It is thought to play a critical role in the visual cycle by functioning as an acceptor of 11-cis-retinol from the isomerohydrolase reaction. The goal here was to evaluate the functional promoter of this gene. METHODS: 5' RACE analysis, promoter-reporter assays, and semiquantitative PCR with exon-specific primers were performed using human-derived RPE cells (ARPE-19 and D407) in culture to evaluate the 5' sequence flanking the RLBP1 gene. In addition, the murine, bovine, and porcine RLBP1 genes were evaluated in silico to identify likely proximal promoter/exon 1 sequences similar to the human gene. RESULTS: 5' RACE analysis revealed the presence of a previously undescribed exon in the RLBP1 gene. This was confirmed by analysis of the GenBank Human EST database, which revealed the presence of 18 sequences matching exon 1. Exon-specific PCR revealed that most CRALBP transcripts expressed in ARPE-19 cells contain both exon 1 and the final exon, suggesting that the primary promoter of CRALBP exists 5' of the newly identified exon 1. Highly homologous sequences in the murine, bovine, and porcine genes were also identified. Finally, promoter-reporter constructs revealed a minimal sequence necessary for promoter function and indicated significantly greater promoter activity compared with previously described RLBP1 promoters. CONCLUSIONS: The findings presented here suggest that CRALBP transcripts in RPE cells contain a noncoding exon in addition to a newly described promoter and, by definition, an additional intron. This finding sets the stage for a mechanistic understanding of the high degree of cell type-specific expression of RLBP1.
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Proteínas Portadoras/genética , Epitelio Pigmentado Ocular/fisiología , Regiones Promotoras Genéticas/genética , Visión Ocular/genética , Región de Flanqueo 5'/genética , Animales , Secuencia de Bases , Bovinos , Línea Celular , Elementos de Facilitación Genéticos/genética , Exones/genética , Humanos , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Epitelio Pigmentado Ocular/citología , PorcinosRESUMEN
In response to growth factors, vascular smooth muscle cells (VSMCs) undergo a phenotypic modulation from a contractile, non-proliferative state to an activated, migratory state. This transition is characterized by changes in their gene expression profile, particularly by a significant down-regulation of contractile proteins. Platelet-derived growth factor (PDGF)-BB has long been known to initiate VSMC de-differentiation and mitogenesis. Insulin-like growth factor (IGF)-I, on the other hand, has differing effects depending on the model studied. Here, we report that both IGF-I and PDGF-BB stimulated VSMC de-differentiation of rat heart-derived SMCs in culture, although only PDGF-BB was capable of inducing proliferation. Although both PDGF-BB and IGF-I stimulation resulted in decreased smooth muscle alpha-actin expression and increased matrix metalloproteinase (MMP)-2 expression, the response to IGF-I was significantly more rapid. The increased MMP-2 expression in response to both growth factors was due to increased transcription rates and was dependent on the action of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. Both PDGF-BB and IGF-I activated PI3K/Akt to similar degrees; however, only PDGF-BB concomitantly stimulated an inhibitory signaling pathway that antagonized the effects of Akt but did not alter the extent or duration of Akt activation. Together, these findings suggest that changes in MMP-2 expression are part of the program of VSMC phenotypic modulation and that both PDGF-BB and IGF-I, despite their different abilities to induce proliferation in this model, are capable of inducing VSMC activation.
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Factor I del Crecimiento Similar a la Insulina/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Músculo Liso Vascular , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/fisiología , Animales , Becaplermina , Biomarcadores/metabolismo , Diferenciación Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Genes Reporteros , Humanos , Metaloproteinasa 2 de la Matriz/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-sis , Ratas , Ratas Endogámicas WKY , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: In a previous study, we reported that keratinocyte growth factor (KGF) produced a rapid increase in the motility of ER-positive breast cancer cells. Others have demonstrated that KGF treatment in rodent species produces rapid mammary ductal hyperplasia. Epithelial cells do not produce KGF; thus, in the present study, MCF-7 cells were stably transfected with a KGF-expressing vector and the motility and morphology of the transfected, non-transfected and empty vector cell lines compared. MATERIALS AND METHODS: A mammalian expression vector containing a KGF cDNA was transfected into MCF-7/beta cells, and two stable clones (MCF-7/beta/KGF-T8 and MCF-7/beta/KGF-T9) were identified. Western blotting of conditioned medium from these clones was used to confirm the expression of KGF. The motility of wild-type and KGF-transfected MCF-7 cells was compared using time-lapse videomicroscopy and a cell culture wounding model which examined cell migration over a period of 1-3 days. RESULTS: The Western blots demonstrated that the expression of KGF in both the MCF-7/beta/KGF-T8 and MCF-7/beta/KGF-T9 cell lines was higher than the wild-type and MCF-7/beta cell lines. The cell proliferation and migration distance was significantly greater for both KGF-transfected MCF-7 cell lines than the wild-type and MCF-7/beta cell lines under the same experimental conditions. Further, changes in motile morphology were observed in both the MCF-7/beta/KGF-T8 and MCF-7/beta/KGF-T9 cell lines. In addition, the MCF-7/beta/KGF-T8 clone was found to produce much larger tumors than both the MCF-7/beta/KGF-T9 and EV clones in mouse xenografts. These results indicated that autocrine production of KGF in the KGF-transfected MCF-7 cell lines enhanced cell migration, migration-related morphology and xenograft tumor growth. CONCLUSION: KGF-transfected MCF-7 cells displayed a much greater motility than non-transfected cells, confirming the KGF motility enhancement effect which we previously reported. The use of KGF-transfected breast cancer cells in the xenograft model may help to study the mechanism of KGF-mediated cell motility and to identify specific KGF antagonists that may be used to prevent or impede KGF-mediated metastatic progression.
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Neoplasias de la Mama/patología , Movimiento Celular/fisiología , Factor 7 de Crecimiento de Fibroblastos/fisiología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Factor 7 de Crecimiento de Fibroblastos/biosíntesis , Factor 7 de Crecimiento de Fibroblastos/genética , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Transfección , Trasplante HeterólogoRESUMEN
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
BACKGROUND AND OBJECTIVES: There exist contradictory reports about low-intensity laser light-stimulated cell proliferation. The purpose of this study was to determine the effect of wavelength on proliferation of cultured murine cells. STUDY DESIGN/MATERIALS AND METHODS: Proliferation of primary cell cultures was measured after irradiation with varying laser wavelengths. RESULTS: Fibroblasts proliferated faster than endothelial cells in response to laser irradiation. Maximum cell proliferation occurred with 665 and 675 nm light, whereas 810 nm light was inhibitory to fibroblasts. CONCLUSIONS: These observations suggest that both wavelength and cell type influence the cell proliferation response to low-intensity laser irradiation.
Asunto(s)
Proliferación Celular/efectos de la radiación , Células Endoteliales/efectos de la radiación , Fibroblastos/efectos de la radiación , Terapia por Luz de Baja Intensidad , Animales , Técnicas de Cultivo de Célula , Relación Dosis-Respuesta en la Radiación , Células Endoteliales/fisiología , Fibroblastos/fisiología , Masculino , Ratones , Ratones Endogámicos C3HRESUMEN
Induced antitumor immunity is a highly effective and long-term cure for cancer, particularly for metastatic tumors. Laser immunotherapy was developed to induce such an immunologic response. It involves intratumoral administration of a light-absorbing dye and a specially formulated immunoadjuvant, followed by noninvasive irradiation of a near-infrared laser. Treatment of DMBA-4 metastatic mammary tumors in rats with this approach has resulted in local control of primary tumors and eradication of untreated distant metastases. After laser immunotherapy, rats were resistant to tumor rechallenge and developed immunity, which could be adoptively transferred. To better understand the immunity induced in this tumor model, immunization using freeze-thaw DMBA-4 cell lysates was performed, followed by tumor challenge 21 days later. Tumor cell lysate immunization delayed the emergence of metastases but did not provide immunity against the tumor challenge. Also performed was surgical resection of primary tumors before the observation of metastatic tumors. Removal of primary tumors was unsuccessful at changing the course of tumor progression. Tumors re-emerged at the primary sites, and metastases developed at multiple remote sites. In contrast, tumor-bearing rats successfully treated by laser immunotherapy experienced tumor regression and eradication and developed strong resistance to repeated challenges by tumor cells of the same type. Our results show that laser immunotherapy could have potential for the treatment of metastatic tumors by inducing tumor-specific, long-lasting immunity.
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Inmunoterapia/métodos , Terapia por Láser , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/radioterapia , 9,10-Dimetil-1,2-benzantraceno , Traslado Adoptivo , Animales , Carcinógenos , Femenino , Liofilización , Neoplasias Mamarias Experimentales/cirugía , Metástasis de la Neoplasia , Ratas , Ratas Endogámicas WFRESUMEN
The ability of cocaine to interact with sigma receptors suggests a viable target for medications development. Recently, numerous novel compounds and antisense oligodeoxynucleotides targeting sigma receptors have been synthesized and shown to prevent the behavioral toxicity and psychomotor stimulant effects of cocaine in animals. Protective doses of sigma receptor antagonists have also been shown to prevent changes in gene expression that are induced by cocaine. Together, the studies provide insight and promising future directions for the development of potential medications for the treatment of cocaine addiction and overdose.
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Cocaína/antagonistas & inhibidores , Sistemas de Liberación de Medicamentos/métodos , Receptores sigma/antagonistas & inhibidores , Tecnología Farmacéutica/métodos , Animales , Cocaína/metabolismo , Humanos , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/farmacología , Receptores sigma/metabolismoRESUMEN
Matrix metalloproteinase (MMP)-2 and MMP-9 are closely related metalloproteinases that are implicated in angiogenesis. The two proteins have a similar domain structure and highly homologous catalytic domains, making them an excellent comparative model for understanding the structural basis of substrate recognition by the MMP family. Although the two MMPs exhibit some overlap in substrate recognition, our recent work showed that MMP-2 can cleave a set of peptide substrates that are only poorly recognized by MMP-9 (Chen, E. I., Kridel, S. J., Howard, E. W., Li, W., Godzik, A., and Smith, J. W. (2002) J. Biol. Chem. 277, 4485-4491). Mutations at the P(2) position of these peptide substrates dramatically reduced their selectivity for MMP-2. Inspection of the corresponding S(2) pocket of the substrate-binding cleft of the protease reveals that MMP-9 contains an Asp, whereas MMP-2 contains Glu. Here, we test the hypothesis that this conservative substitution has a role in substrate selectivity. Mutation of Glu(412) in MMP-2 to Asp significantly reduced the hydrolysis of selective substrates, with only a minor effect on hydrolysis of non-selective substrates. The predominant effect of the mutation is at the level of k(cat), or turnover rate, with reductions reaching as high as 37-fold. The residues that occupy this position in other MMPs are highly variable, providing a potential structural basis for substrate recognition across the MMP family.