RESUMEN
Despite expanded definition of the leukocyte adhesion cascade and mechanisms underlying individual steps, very little is known about regulatory mechanisms controlling sequential shifts between steps. We tested the hypothesis that metalloproteinases provide a mechanism to rapidly transition monocytes between different steps. Our study identifies diapedesis as a step targeted by metalloproteinase activity. Time-lapse video microscopy shows that the presence of a metalloproteinase inhibitor results in a doubling of the time required for human monocytes to complete diapedesis on unactivated or inflamed human endothelium, under both static and physiological-flow conditions. Thus, diapedesis is promoted by metalloproteinase activity. In contrast, neither adhesion of monocytes nor their locomotion over the endothelium is altered by metalloproteinase inhibition. We further demonstrate that metalloproteinase inhibition significantly elevates monocyte cell surface levels of integrins CD11b/CD18 (Mac-1), specifically during transendothelial migration. Interestingly, such alterations are not detected for other endothelial- and monocyte-adhesion molecules that are presumed metalloproteinase substrates. Two major transmembrane metalloproteinases, a disintegrin and metalloproteinase (ADAM)17 and ADAM10, are identified as enzymes that control constitutive cleavage of Mac-1. We further establish that knockdown of monocyte ADAM17, but not endothelial ADAM10 or ADAM17 or monocyte ADAM10, reproduces the diapedesis delay observed with metalloproteinase inhibition. Therefore, we conclude that monocyte ADAM17 facilitates the completion of transendothelial migration by accelerating the rate of diapedesis. We propose that the progression of diapedesis may be regulated by spatial and temporal cleavage of Mac-1, which is triggered upon interaction with endothelium.
Asunto(s)
Proteínas ADAM/fisiología , Metaloproteasas/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Migración Transendotelial y Transepitelial/inmunología , Proteínas ADAM/deficiencia , Proteínas ADAM/metabolismo , Proteína ADAM17 , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana , Humanos , Antígeno de Macrófago-1/metabolismo , Metaloproteasas/antagonistas & inhibidores , Monocitos/enzimología , Especificidad por Sustrato/inmunología , Imagen de Lapso de Tiempo/métodosRESUMEN
BACKGROUND: Extremity trauma is the most common injury seen in combat hospitals as well as in civilian trauma centers. Major skeletal muscle injuries that are complicated by ischemia often result in substantial muscle loss, residual disability, or even amputation, yet few treatment options are available. A therapy that would increase skeletal muscle tolerance to hypoxic damage could reduce acute myocyte loss and enhance preservation of muscle mass in these situations. QUESTIONS/PURPOSES: In these experiments, we investigated (1) whether cobalt protoporphyrin (CoPP), a pharmacologic inducer of cytoprotective heme oxygenase-1 (HO-1), would upregulate HO-1 expression and activity in skeletal muscle, tested in muscle-derived stem cells (MDSCs); and (2) whether CoPP exposure would protect MDSCs from cell death during in vitro hypoxia/reoxygenation. Then, using an in vivo mouse model of hindlimb ischemia/reperfusion injury, we examined (3) whether CoPP pharmacotherapy would reduce skeletal muscle damage when delivered after injury; and (4) whether it would alter the host inflammatory response to injury. METHODS: MDSCs were exposed in vitro to a single dose of 25 µΜ CoPP and harvested over 24 to 96 hours, assessing HO-1 protein expression by Western blot densitometry and HO-1 enzyme activity by cGMP levels. To generate hypoxia/reoxygenation stress, MDSCs were treated in vitro with phosphate-buffered saline (vehicle), CoPP, or CoPP plus an HO-1 inhibitor, tin protoporphyrin (SnPP), and then subjected to 5 hours of hypoxia (< 0.5% O2) followed by 24 hours of reoxygenation and evaluated for apoptosis. In vivo, hindlimb ischemia/reperfusion injury was produced in mice by unilateral 2-hour tourniquet application followed by 24 hours of reperfusion. In three postinjury treatment groups (n = 7 mice/group), CoPP was administered intraperitoneally during ischemia, at the onset of reperfusion, or 1 hour later. Two control groups of mice with the same injury received phosphate-buffered saline (vehicle) or the HO-1 inhibitor, SnPP. Myocyte damage in the gastrocnemius and tibialis anterior muscles was determined by uptake of intraperitoneally delivered Evans blue dye (EBD), quantified by image analysis. On serial sections, inflammation was gauged by the mean myeloperoxidase staining intensity per unit area over the entirety of each muscle. RESULTS: In MDSCs, a single exposure to CoPP increased HO-1 protein expression and enzyme activity, both of which were sustained for 96 hours. CoPP treatment of MDSCs reduced apoptotic cell populations by 55% after in vitro hypoxia/reoxygenation injury (from a mean of 57.3% apoptotic cells in vehicle-treated controls to 25.7% in CoPP-treated cells, mean difference 31.6%; confidence interval [CI], 28.1-35.0; p < 0.001). In the hindlimb ischemia/reperfusion model, CoPP delivered during ischemia produced a 38% reduction in myocyte damage in the gastrocnemius muscle (from 86.4% ± 7% EBD(+) myofibers in vehicle-treated, injured controls to 53.2% EBD(+) in CoPP-treated muscle, mean difference 33.2%; 95% CI, 18.3, 48.4; p < 0.001). A 30% reduction in injury to the gastrocnemius was seen with drug delivery at the onset of reperfusion (to 60.6% ± 13% EBD(+) with CoPP treatment, mean difference 25.8%; CI, 12.2-39.4; p < 0.001). In the tibialis anterior, however, myocyte damage was decreased only when CoPP was given at the onset of reperfusion, resulting in a 27% reduction in injury (from 78.8% ± 8% EBD(+) myofibers in injured controls to 58.3% ± 14% with CoPP treatment, mean difference 20.5%; CI, 6.1-35.0; p = 0.004). Delaying CoPP delivery until 1 hour after tourniquet release obviated the protective effect in both muscles. Mean MPO staining intensity per unit area, indicating the host inflammatory response, decreased by 27-34% across both the gastrocnemius and tibialis anterior muscles when CoPP was given either during ischemia or at the time of reperfusion. Delaying drug delivery until 1 hour after the start of reperfusion abrogated this antiinflammatory effect. CONCLUSIONS: CoPP can decrease skeletal muscle damage when given early in the course of ischemia/reperfusion injury and also provide protection for regenerative stem cell populations. CLINICAL RELEVANCE: Pharmacotherapy with HO-1 inducers, delivered in the field, on hospital arrival, or during trauma surgery, may improve preservation of muscle mass and muscle-inherent stem cells after severe ischemic limb injury.
Asunto(s)
Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Protoporfirinas/farmacología , Daño por Reperfusión/prevención & control , Células Madre/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Citoprotección , Modelos Animales de Enfermedad , Inducción Enzimática , Inhibidores Enzimáticos/farmacología , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hemo-Oxigenasa 1/biosíntesis , Miembro Posterior , Mediadores de Inflamación/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/biosíntesis , Ratones Endogámicos C57BL , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Regeneración/efectos de los fármacos , Daño por Reperfusión/enzimología , Daño por Reperfusión/patología , Células Madre/enzimología , Células Madre/patología , Factores de TiempoRESUMEN
An improved understanding of the factors that regulate the migration of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) would provide new insights into human heart development and suggest novel strategies to improve their electromechanical integration after intracardiac transplantation. Since nothing has been reported as to the factors controlling hESC-CM migration, we hypothesized that hESC-CMs would migrate in response to the extracellular matrix and soluble signaling molecules previously implicated in heart morphogenesis. To test this, we screened candidate factors by transwell assay for effects on hESC-CM motility, followed by validation via live-cell imaging and/or gap-closure assays. Fibronectin (FN) elicited a haptotactic response from hESC-CMs, with cells seeded on a steep FN gradient showing nearly a fivefold greater migratory activity than cells on uniform FN. Studies with neutralizing antibodies indicated that adhesion and migration on FN are mediated by integrins α-5 and α-V. Next, we screened 10 soluble candidate factors by transwell assay and found that the noncanonical Wnt, Wnt5a, elicited an approximately twofold increase in migration over controls. This effect was confirmed using the gap-closure assay, in which Wnt5a-treated hESC-CMs showed approximately twofold greater closure than untreated cells. Studies with microfluidic-generated Wnt5a gradients showed that this factor was chemoattractive as well as chemokinetic, and Wnt5a-mediated responses were inhibited by the Frizzled-1/2 receptor antagonist, UM206. In summary, hESC-CMs show robust promigratory responses to FN and Wnt5a, findings that have implications on both cardiac development and cell-based therapies.
Asunto(s)
Células Madre Embrionarias/citología , Matriz Extracelular/efectos de los fármacos , Fibronectinas/farmacología , Miocitos Cardíacos/efectos de los fármacos , Proteínas Proto-Oncogénicas/farmacología , Proteínas Wnt/farmacología , Anticuerpos Neutralizantes/farmacología , Adhesión Celular , Diferenciación Celular , Movimiento Celular/efectos de los fármacos , Cámaras de Difusión de Cultivos , Células Madre Embrionarias/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Expresión Génica , Humanos , Imagen Molecular , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Fibronectina/antagonistas & inhibidores , Receptores de Fibronectina/genética , Receptores de Fibronectina/metabolismo , Transducción de Señal , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5aRESUMEN
The differentiation of pluripotent stem cells involves transition through a series of specific cell states. To understand these cell fate decisions, the field needs improved genetic tools for the labeling, lineage tracing and selection of specific cell types from heterogeneous differentiating populations, particularly in the human embryonic stem cell (hESC) system. We used zinc finger nuclease technology to stably insert a unique, selectable, floxed dual-fluorescence reporter transgene into the AAVS1 locus of RUES2 hESCs. This "stoplight" transgene, mTmG-2a-Puro, strongly expresses membrane-localized tdTomato red fluorescent protein until Cre-dependent recombination causes a switch to expression of membrane-localized enhanced green fluorescent protein (eGFP) and puromycin resistance. First, to validate this system in undifferentiated cells, we transduced transgenic hESCs with a lentiviral vector driving constitutive expression of Cre and observed the expected phenotypic switch. Next, to demonstrate its utility in lineage-specific selection, we transduced differentiated cultures with a lentiviral vector in which the striated muscle-specific CK7 promoter drives Cre expression. This yielded near-homogenous populations of eGFP(+) hESC-derived cardiomyocytes. The mTmg-2a-Puro hESC line described here represents a useful new tool for both in vitro fate mapping studies and the selection of useful differentiated cell types.
Asunto(s)
Células Madre Embrionarias/metabolismo , Marcación de Gen/métodos , Ingeniería Genética/métodos , Proteínas Luminiscentes/metabolismo , Transgenes/genética , Diferenciación Celular/genética , Células Cultivadas , Células Madre Embrionarias/citología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lentivirus/genética , Proteínas Luminiscentes/genética , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Factores de Tiempo , Dedos de Zinc/genéticaRESUMEN
BACKGROUND: Hundreds of genes, including muscle creatine kinase (MCK), are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood. RESULTS: Modulatory region 1 (MR1) is a 1-kb regulatory region within MCK intron 1 that is highly active in terminally differentiating skeletal myocytes in vitro. A MCK small intronic enhancer (MCK-SIE) containing a paired E-box/myocyte enhancer factor 2 (MEF2) regulatory motif resides within MR1. The SIE's transcriptional activity equals that of the extensively characterized 206-bp MCK 5'-enhancer, but the MCK-SIE is flanked by regions that can repress its activity via the individual and combined effects of about 15 different but highly conserved 9- to 24-bp sequences. ChIP and ChIP-Seq analyses indicate that the SIE and the MCK 5'-enhancer are occupied by MyoD, myogenin and MEF2. Many other E-boxes located within or immediately adjacent to intron 1 are not occupied by MyoD or myogenin. Transgenic analysis of a 6.5-kb MCK genomic fragment containing the 5'-enhancer and proximal promoter plus the 3.2-kb intron 1, with and without MR1, indicates that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers (types I and IIa, respectively), but is not required for expression in fast-twitch muscle fibers (types IIb and IId). CONCLUSIONS: In this study, we discovered that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers and that MR1's positive transcriptional activity depends on a paired E-box MEF2 site motif within a SIE. This is the first study to delineate the DNA controls for MCK expression in different skeletal muscle fiber types.
RESUMEN
Vertebrate precardiac mesoderm contains cells destined to become cardiomyocyte or endothelial cells. To determine the stability of these phenotypes freshly isolated embryonic day (E) 2.5-E6 chicken hearts were immunostained for myosin heavy chain (MyHC) to identify cardiomyocytes, and von Willebrand factor (vWF) and Flk-1 to identify endothelial cells. At E2.5-E3, 90% of cells express only MyHC and 6% express only vWF/Flk-1. However, 2% MyHC+ cells in E2.5-E3 hearts and 0.3% in E4-E6 hearts, also express vWF/Flk-1; and when cultured 3 days, >40% of the MyHC+ cells express vWF/Flk-1, but they do not express Vezf1, vascular endothelial cadherin, or Tie2. Thus, only a subset of endothelial genes are induced in cultured cardiomyocytes. While the subsequent developmental fate of embryonic heart cells exhibiting a vWF+/MyHC+ phenotype is unknown, analysis of this phenotype may provide information pertinent to mechanisms of cell phenotype stability, cellular transdifferentiation, and induction of stable cell types from embryonic stem cells.
Asunto(s)
Biología Evolutiva/métodos , Células Endoteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Miocitos Cardíacos/metabolismo , Animales , Células Cultivadas , Embrión de Pollo/fisiología , Técnica del Anticuerpo Fluorescente Indirecta , Inmunohistoquímica/métodos , Modelos Biológicos , Modelos Genéticos , Miocardio/metabolismo , Factores de TiempoRESUMEN
It was recently reported that human umbilical endothelial vein cells (HUVECs) transdifferentiate and express cardiac genes when co-cultured with rat neonatal cardiomyocytes (Condorelli et al. (2001)). If substantiated and optimized, this phenomenon could have many therapeutic applications. We re-investigated the HUVEC-rat neonatal cardiomyocyte co-culture system but detected cardiomyocyte markers (sarcomeric myosin) in only 1.2% of the cells containing nuclei that were immuno-positive for human nuclear antigen (HNA); and the frequency of such cells was not enhanced in co-cultures containing more embryonic cardiomyocytes. Because the majority of HNA-positive/myosin-positive cells were binucleated, we tested the hypothesis that these cells resulted from HUVEC-cardiomyocyte fusion rather than from HUVEC transdifferentiation. Analysis with a Cre/lox recombination assay indicated that virtually all HUVECs containing cardiac markers had fused with cardiomyocytes. To determine whether human cardiomyocyte genes are activated at low levels in HUVEC-cardiomyocyte co-cultures, quantitative RT-PCR was performed with primers specific for human beta-MyHC and cTnI. We found no evidence for transcriptional activation of these genes. None of our data support conversion of HUVECs to cardiomyocytes.
Asunto(s)
Células Endoteliales/fisiología , Regulación de la Expresión Génica , Miocitos Cardíacos/fisiología , Venas Umbilicales/fisiología , Animales , Fusión Celular/métodos , Células Cultivadas , Células Endoteliales/citología , Humanos , Miocitos Cardíacos/citología , Especificidad de Órganos/fisiología , Ratas , Ratas Sprague-Dawley , Venas Umbilicales/citologíaRESUMEN
Mouse P19 embryonal carcinoma cells undergo cardiogenesis in response to high density and DMSO. We have derived a clonal subline that undergoes cardiogenesis in response to high density, but without requiring exposure to DMSO. The new subline retains the capacity to differentiate into skeletal muscle and neuronal cells in response to DMSO and retinoic acid. However, upon aggregation, these Oct 4-positive cells, termed P19-SI because they "self-induce" cardiac muscle, exhibit increased mRNAs encoding the mesodermal factor Brachyury, cardiac transcription factors Nkx 2.5 and GATA 4, the transcriptional repressor Msx-1, and cytokines Wnt 3a, Noggin, and BMP 4. Exposure of aggregated P19-SI cells to BMP 4, a known inducer of cardiogenesis, accelerates cardiogenesis, as determined by rhythmic beating and myosin staining. However, cardiogenesis is severely inhibited when P19-SI cells are aggregated in the presence of BMP 4. These results demonstrate that cell-cell interaction is required before P19-SI cells can undergo a cardiogenic response to BMP 4. A concurrent increase in the expression of Msx-1 suggests one possible process underlying the inhibition of cardiogenesis. The phenotype of P19-SI cells offers an opportunity to explore new aspects of cardiac induction.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Comunicación Celular , Diferenciación Celular , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Animales , Biomarcadores , Proteína Morfogenética Ósea 4 , Diferenciación Celular/efectos de los fármacos , Forma de la Célula , Células Cultivadas , Dimetilsulfóxido/farmacología , Endodermo/metabolismo , Endodermo/patología , Regulación de la Expresión Génica , Factor de Transcripción MSX1/metabolismo , Ratones , Miocitos Cardíacos/efectos de los fármacos , Fenotipo , Transducción de Señal , Factores de Tiempo , Tretinoina/farmacologíaRESUMEN
Using the COS cell transfection assay developed previously, we examined which domains of myosin-binding proteins C and H (MyBP-C and MyBP-H) are involved in intracellular interactions with sarcomeric myosin heavy chain (MyHC). Earlier studies demonstrated that overexpression of sarcomeric MyHC in COS cells results in the cytoplasmic assembly of anisotropic, spindle-like aggregates of myosin-containing filaments in the absence of other myofibrillar proteins. When the same sarcomeric MyHC was co-expressed with either MyBP-C or MyBP-H, prominent cable-like co-polymers of MyHC and the MyBPs formed in the cytoplasm instead of the spindle-like aggregates formed by MyHC alone. In vitro binding assays have shown that the C-terminal IgI domain of both MyBP-C (domain C10) and MyBP-H (domain H4) contains the light meromyosin (LMM)-binding sites of each molecule, but this domain cannot explain all of the intracellular properties of the molecules. For example, domains C7-C10 of MyBP-C and domains H1-H4 of MyBP-H are required for the faithful targeting of these proteins to the A-bands of myofibrils in skeletal muscle. Using truncation mutants of both MyBPs tagged with either green fluorescent protein (GFP) or c-myc, we now demonstrate that the last four domains of both MyBP-C and MyBP-H colocalize with the full-length proteins in the MyHC/MyBP cable polymers when co-transfected with MyHC in COS cells. Deletion of the C-terminal IgI domain in either MyBP-C or MyBP-H abrogated cable formation, but the expressed proteins could still colocalize with MyHC-containing filament aggregates. Co-expression of only the C-terminal IgI domain of MyBP-C with sarcomeric MyHC was sufficient for cable formation and colocalization with myosin. We conclude that the C-terminal IgI domains of both MyBP-H and MyBP-C are both necessary and sufficient for inducing MyHC/MyBP cable formation in this COS cell system. However, there must be other myosin-binding sites in MyBP-C and MyBP-H that explain the co-distribution of these proteins with myosin filaments in the absence of cable formation. These latter sites are neither sufficient nor required for cable formation.