RESUMEN
OBJECTIVES: To develop a sustainable business model for pharmacist-provided comprehensive medication management services in a patient-centered medical home. Secondarily, to evaluate the impact that the pharmacist had on clinical (glycosylated hemoglobin [A1C], low-density lipoprotein [LDL], and blood pressure) and economic (physician productivity and cost avoidance) outcomes. PRACTICE DESCRIPTION: This pilot project took place at the Palmetto Primary Care Physicians Trident office in North Charleston, South Carolina, from October 2013 to September 2014. At the time, the practice employed 5 physicians and 2 nurse practitioners and served more than 20,000 patients. PRACTICE INNOVATION: The pharmacist targeted patients with diabetes, lipid disorders, hypertension, congestive heart failure, obesity, polypharmacy, and treatment regimen nonadherence for his comprehensive medication management services. The pharmacist was available for immediate consultation or referrals by appointment 5 days per week. Services provided by the pharmacist were billed as medication therapy management or "incident to" physician evaluation and management services codes. EVALUATION: Number of patients seen per day, revenue collected from services rendered by the pharmacist, physician productivity and payment, cost avoidance, and health quality metrics (A1C, LDL, and blood pressure) were measured to determine the financial sustainability and clinical impact of the project. RESULTS AND IMPLICATIONS: The pharmacist was able to see an average of 11 patients per day, which was 72% of his capacity. The practice collected about $7400 per month for services rendered by the pharmacist. The average daily payment for services rendered by the physicians in the practice increased by 20.6%. More than 70% of uncontrolled patients had an improvement in clinical outcomes, such as A1C, LDL, and blood pressure. CONCLUSION: This project demonstrates the sustainable business model for embedding a pharmacist into a patient-centered medical home.
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Administración del Tratamiento Farmacológico/organización & administración , Atención Dirigida al Paciente/organización & administración , Servicios Farmacéuticos/organización & administración , Farmacéuticos/organización & administración , Presión Sanguínea/fisiología , LDL-Colesterol/sangre , Hemoglobina Glucada/análisis , Humanos , Administración del Tratamiento Farmacológico/economía , Modelos Organizacionales , Atención Dirigida al Paciente/economía , Servicios Farmacéuticos/economía , Farmacéuticos/economía , Proyectos Piloto , Atención Primaria de Salud/economía , Atención Primaria de Salud/organización & administración , Rol Profesional , South CarolinaRESUMEN
BACKGROUND: The nematode Caenhorhabditis elegans offers great power for the identification and characterization of genes that regulate behavior. In support of this effort, analytical methods are required that provide dimensional analyses of subcomponents of behavior. Previously, we demonstrated that loss of the presynaptic dopamine (DA) transporter, dat-1, evokes DA-dependent Swimming-Induced Paralysis (Swip) (Mcdonald et al., 2007), a behavior compatible with forward genetic screens (Hardaway et al., 2012). NEW METHOD: Here, we detail the development and implementation of SwimR, a set of tools that provide for an automated, kinetic analysis of C. elegans Swip. SwimR relies on open source programs that can be freely implemented and modified. RESULTS: We show that SwimR can display time-dependent alterations of swimming behavior induced by drug-treatment, illustrating this capacity with the dat-1 blocker and tricyclic antidepressant imipramine (IMI). We demonstrate the capacity of SwimR to extract multiple kinetic parameters that are impractical to obtain in manual assays. COMPARISON WITH EXISTING METHODS: Standard measurements of C. elegans swimming utilizes manual assessments of the number of animals exhibiting swimming versus paralysis. Our approach deconstructs the time course and rates of movement in an automated fashion, offering a significant increase in the information that can be obtained from swimming behavior. CONCLUSIONS: The SwimR platform is a powerful tool for the deconstruction of worm thrashing behavior in the context of both genetic and pharmacological manipulations that can be used to segregate pathways that underlie nematode swimming mechanics.
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Parálisis/diagnóstico , Parálisis/etiología , Natación , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Antidepresivos Tricíclicos/farmacología , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Relación Dosis-Respuesta a Droga , Procesamiento Automatizado de Datos , Imipramina/farmacología , Oxigenasas de Función Mixta/genética , Mutación/genética , Parálisis/inducido químicamente , Parálisis/genética , Receptores de Dopamina D2/genéticaRESUMEN
Advances in understanding of the maintenance of the cardiac valves during normal cardiac function and response to injury have led to several novel findings, including that there is contribution of extra-cardiac cells to the major cellular population of the valve: the valve interstitial cell (VIC). While suggested to occur in human heart studies, we have been able to experimentally demonstrate, using a mouse model, that cells of bone marrow hematopoietic stem cell origin engraft into the valves and synthesize collagen type I. Based on these initial findings, we sought to further characterize this cell population in terms of its similarity to VICs and begin to elucidate its contribution to valve homeostasis. To accomplish this, chimeric mice whose bone marrow was repopulated with enhanced green fluorescent protein (EGFP) expressing total nucleated bone marrow cells were used to establish a profile of EGFP(+) valve cells in terms of their expression of hematopoietic antigens, progenitor markers, fibroblast- and myofibroblast-related molecules, as well as their distribution within the valves. Using this profile, we show that normal (non-irradiated, non-transplanted) mice have BM-derived cell populations that exhibit identical morphology and phenotype to those observed in transplanted mice. Collectively, our findings establish that the engraftment of bone marrow-derived cells occurs as part of normal valve homeostasis. Further, our efforts demonstrate that the use of myeloablative irradiation, which is commonly employed in studies involving bone marrow transplantation, does not elicit changes in the bone marrow-derived VIC phenotype in recipient mice.
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Células de la Médula Ósea/citología , Válvulas Cardíacas/citología , Válvulas Cardíacas/metabolismo , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Femenino , Glicoproteínas/metabolismo , Válvulas Cardíacas/efectos de la radiación , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Homeostasis , Antígenos Comunes de Leucocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Péptidos/metabolismo , Fenotipo , Irradiación Corporal TotalRESUMEN
We evaluated the self-assembly properties of uniluminal vascular spheroids having outer layers of vascular smooth muscle cells and a contiguous inner layer of endothelial cells lining a central lumen. We showed that while pairs of uniluminal vascular spheroids suspended in culture medium fused to form a larger diameter spheroidal structure, spheroids in collagen hydrogels formed elongated structures. These findings highlight the potential use of uniluminal vascular spheroids as modules to engineer blood vessels. We also demonstrate that uniluminal vascular spheroid fusion conforms to models describing the coalescence of liquid drops. Furthermore, the fusion of uniluminal vascular spheroids in vitro closely resembled the in vivo process by which the descending aorta forms from the fusion of the paired dorsal aortae during embryonic development. Together, the findings indicate that tissue liquidity underlies uniluminal vascular spheroid fusion and that in vivo anastomosis of blood vessels may involve a similar mechanism.
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Vasos Sanguíneos/embriología , Esferoides Celulares/fisiología , Animales , Aorta/embriología , Fusión Celular , Colágeno , Femenino , Hidrogeles , Ratones , Modelos Cardiovasculares , Embarazo , Conejos , Ratas , Ingeniería de TejidosRESUMEN
Embryonic mouse allantoic tissue (E8.5) was cultured in hanging drops to generate a three-dimensional vascular micro-tissue. The resulting tissue spheroids had an inner network of small diameter vessels expressing platelet endothelial cell adhesion molecule-1 (PECAM-1) and an outer layer of cells expressing SMalphaA, SM22-alpha, and SM-MHC. In a subsequent phase of culture, the fusion-promoting activity of vascular endothelial growth factor (VEGF) was used to transform the inner network of small diameter endothelial tubes into a contiguous layer of cells expressing PECAM-1, CD34, and VE-cadherin that circumscribed a central lumen-like cavity. The blood vessel-like character of the VEGF-treated spheroids was further demonstrated by their physiologically relevant vasodilatory and contractile responses, including contraction induced by KCl and relaxation stimulated by high-density lipoproteins and acetylcholine-induced nitric oxide production.
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Neovascularización Fisiológica/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Actinas/metabolismo , Alantoides/irrigación sanguínea , Alantoides/efectos de los fármacos , Alantoides/embriología , Alantoides/metabolismo , Animales , Células Endoteliales/metabolismo , Antígenos de Histocompatibilidad/metabolismo , Ratones , Ratones Endogámicos ICRRESUMEN
OBJECTIVES: Pancreatic acinar cells and hepatocytes arise from the same cell population located within the embryonic endoderm. It has been reported that a multipotent population of liver cells is capable of differentiating into pancreatic cells. Recent studies revealed that murine and human hematopoietic cells could generate hepatocytes in vivo. Based on this developmental proximity between liver and pancreatic acinar cells, we examined whether human cord blood (CB) cells can generate pancreatic cells in vivo using a murine xenograft model. METHODS: We transplanted 1 x 10 CD34 human CB cells into "conditioned" newborn nonobese diabetic-severe combined immunodeficiency/beta-2 microglobulin-null mice via facial vein injection and, 3 to 4 months later, examined the pancreata from recipient mice showing high-level human multilineage hematopoietic engraftment in the bone marrow. RESULTS: Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses revealed human amylase mRNA and protein expression, respectively, in the pancreata from recipient mice. Using fluorescence in situ hybridization, we identified human alpha-satellite, DNA-positive cells with a morphology characteristic of pancreatic acinar cells. We also identified cells in paraffin sections of the pancreata that expressed amylase mRNA, had morphological characteristics of acinar cells, and contained human but not mouse centromeric DNA. CONCLUSION: These findings establish that human umbilical CB cells are capable of generating pancreatic acinar cells via a nonfusion mechanism.
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Amilasas/metabolismo , Diferenciación Celular , Trasplante de Células Madre de Sangre del Cordón Umbilical , Sangre Fetal/citología , Páncreas Exocrino/citología , Células Madre , Amilasas/genética , Animales , Animales Recién Nacidos , Antígenos CD34/análisis , Forma de la Célula , ADN Satélite/metabolismo , Sangre Fetal/enzimología , Sangre Fetal/inmunología , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Páncreas Exocrino/enzimología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/enzimología , Células Madre/inmunología , Factores de Tiempo , Trasplante Heterólogo , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMEN
To identify genes important to the process of vasculogenesis, we have used a novel meta-analysis approach to evaluate retrospectively the embryonic vascular anomalies observed in over 100 mouse gene knockout studies. Through application of this method, termed Approach for Ranking of Embryonic Vascular Anomalies (AREVA), 12 genes were determined to be critical to vasculogenesis. Importantly, when the 12 genes were considered with respect to VEGF-VEGFR signalling, an integrated network centreing on the ShcA/Ras/Raf/Mek/Erk pathway became apparent. Herein, we discuss how the 12 vasculogenesis-critical genes influence specific stages in the process of vasculogenesis.
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Neovascularización Fisiológica/genética , Animales , Vasos Sanguíneos/citología , Vasos Sanguíneos/embriología , Linaje de la Célula , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Células Endoteliales/citología , Ratones , Seudópodos/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Cubilin is a peripheral membrane protein that interacts with the integral membrane proteins megalin and amnionless to mediate ligand endocytosis by absorptive epithelia such as the extraembryonic visceral endoderm (VE). RESULTS: Here we report the effects of the genetic deletion of cubilin on mouse embryonic development. Cubilin gene deletion is homozygous embryonic lethal with death occurring between 7.5-13.5 days post coitum (dpc). Cubilin-deficient embryos display developmental retardation and do not advance morphologically beyond the gross appearance of wild-type 8-8.5 dpc embryos. While mesodermal structures such as the allantois and the heart are formed in cubilin mutants, other mesoderm-derived tissues are anomalous or absent. Yolk sac blood islands are formed in cubilin mutants but are unusually large, and the yolk sac blood vessels fail to undergo remodeling. Furthermore, somite formation does not occur in cubilin mutants. Morphological abnormalities of endoderm occur in cubilin mutants and include a stratified epithelium in place of the normally simple columnar VE epithelium and a stratified cuboidal epithelium in place of the normally simple squamous epithelium of the definitive endoderm. Cubilin-deficient VE is also functionally defective, unable to mediate uptake of maternally derived high-density lipoprotein (HDL). CONCLUSION: In summary, cubilin is required for embryonic development and is essential for the formation of somites, definitive endoderm and VE and for the absorptive function of VE including the process of maternal-embryo transport of HDL.
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Desarrollo Embrionario , Endodermo/citología , Endodermo/metabolismo , Receptores de Superficie Celular/fisiología , Somitos/fisiología , Animales , Embrión de Mamíferos/anomalías , Exones , Genes Letales , Lipoproteínas HDL/metabolismo , Mesodermo/citología , Ratones , Ratones Noqueados , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Eliminación de Secuencia , Saco Vitelino/irrigación sanguíneaRESUMEN
Cholinergic neurotransmission supports motor, autonomic, and cognitive function and is compromised in myasthenias, cardiovascular diseases, and neurodegenerative disorders. Presynaptic uptake of choline via the sodium-dependent, hemicholinium-3-sensitive choline transporter (CHT) is believed to sustain acetylcholine (ACh) synthesis and release. Analysis of this hypothesis in vivo is limited in mammals because of the toxicity of CHT antagonists and the early postnatal lethality of CHT-/- mice (Ferguson et al., 2004). In Caenorhabditis elegans, in which cholinergic signaling supports motor activity and mutant alleles impacting ACh secretion and response can be propagated, we investigated the contribution of CHT (CHO-1) to facets of cholinergic neurobiology. Using the cho-1 promoter to drive expression of a translational, green fluorescent protein-CHO-1 fusion (CHO-1:GFP) in wild-type and kinesin (unc-104) mutant backgrounds, we establish in the living nematode that the transporter localizes to cholinergic synapses, and likely traffics on synaptic vesicles. Using embryonic primary cultures, we demonstrate that CHO-1 mediates hemicholinium-3-sensitive, high-affinity choline uptake that can be enhanced with depolarization in a Ca(2+)-dependent manner supporting ACh synthesis. Although homozygous cho-1 null mutants are viable, they possess 40% less ACh than wild-type animals and display stress-dependent defects in motor activity. In a choline-free liquid environment, cho-1 mutants demonstrate premature paralysis relative to wild-type animals. Our findings establish a requirement for presynaptic choline transport activity in vivo in a model amenable to a genetic dissection of CHO-1 regulation.
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Acetilcolina/biosíntesis , Proteínas de Transporte de Membrana/fisiología , Actividad Motora/fisiología , Sinapsis/metabolismo , Adaptación Fisiológica , Animales , Animales Modificados Genéticamente , Unión Competitiva , Transporte Biológico/efectos de los fármacos , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Células Cultivadas , Colina/farmacocinética , Colinérgicos/farmacología , Electrofisiología , Eliminación de Gen , Proteínas Fluorescentes Verdes/genética , Hemicolinio 3/farmacología , Proteínas de Transporte de Membrana/deficiencia , Proteínas de Transporte de Membrana/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Distribución TisularRESUMEN
Recent studies evaluating hematopoietic stem cell (HSC) potential raise the possibility that, in addition to embryonic sources, adult valve fibroblasts may be derived from HSCs. To test this hypothesis, we used methods that allow the potential of a single HSC to be evaluated in vivo. This was achieved by isolation and clonal expansion of single lineage-negative (Lin-), c-kit(+), Sca-1(+), CD34- cells from the bone marrow of mice that ubiquitously express enhanced green fluorescent protein (EGFP) combined with transplantation of individual clonal populations derived from these candidate HSCs into a lethally irradiated congenic non-EGFP mouse. Histological analyses of valve tissue from clonally engrafted recipient mice revealed the presence of numerous EGFP+ cells within host valves. A subpopulation of these cells exhibited synthetic properties characteristic of fibroblasts, as evidenced by their expression of mRNA for procollagen 1alpha1. Further, we show by Y-chromosome-specific fluorescence in situ hybridization analysis of female-to-male transplanted mice that the EGFP+ valve cells are the result of HSC-derived cell differentiation and not the fusion of EGFP+ donor cells with host somatic cells. Together, these findings demonstrate HSC contribution to the adult valve fibroblast population.
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Fibroblastos/citología , Válvulas Cardíacas/citología , Trasplante de Células Madre Hematopoyéticas , Animales , Diferenciación Celular , Colágeno Tipo I/genética , Femenino , Proteínas Fluorescentes Verdes/genética , Hematopoyesis , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/análisisRESUMEN
OBJECTIVE: Recent studies have reported that bone marrow cells can give rise to tissue fibroblasts. However, the bone marrow cell(s) that gives rise to fibroblasts has not yet been identified. In the present study, we tested the hypothesis that tissue fibroblasts are derived from hematopoietic stem cells (HSCs) in vivo. METHODS: These studies were conducted using mice whose hematopoiesis had been reconstituted by transplantation of a clonal population of cells derived from a single enhanced green fluorescent protein (EGFP)-positive HSC in conjunction with murine tumor models. RESULTS: When tumors propagated in the transplanted mice were evaluated for the presence of EGFP(+) HSC-derived cells, two prominent populations of EGFP(+) cells were found. The first were determined to be fibroblasts within the tumor stromal capsule, a subset of which expressed type I collagen mRNA and alpha-smooth muscle actin. The second population was a perivascular cell associated with the CD31(+) tumor blood vessels. CONCLUSION: These in vivo findings establish an HSC origin of fibroblasts.
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Fibroblastos/metabolismo , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Neoplasias/metabolismo , Actinas/metabolismo , Animales , Línea Celular Tumoral , Células Clonales , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Fibroblastos/patología , Regulación Neoplásica de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos C57BL , Neoplasias/irrigación sanguínea , Neoplasias/patología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células del Estroma/patología , Trasplante Homólogo , Células Tumorales Cultivadas , Irradiación Corporal TotalRESUMEN
OBJECTIVE: Using transplantation of a clonal population of cells derived from a single hematopoietic stem cell (HSC) of transgenic enhanced green fluorescent protein (EGFP) mice, we have documented the hematopoietic origin of myofibroblasts, such as kidney mesangial cells and brain microglial cells. Because myofibroblasts are thought to be an activated form of fibroblasts, we tested the hypothesis that fibroblasts are derived from HSCs. MATERIALS AND METHODS: Clones of cells derived from single cells of EGFP Ly-5.2 C57Bl/6 mice were transplanted into lethally irradiated Ly-5.1 mice. Using bone marrow and peripheral blood cells from mice showing high-level multilineage hematopoietic reconstitution, we induced growth of fibroblasts in vitro. RESULTS: Culture of EGFP(+) bone marrow cells from clonally engrafted mice revealed adherent cells with morphology typical of fibroblasts. Flow cytometric analysis revealed that the majority of these cells are CD45(-) and express collagen-I and the collagen receptor, discoidin domain receptor 2 (DDR2). Reverse transcriptase polymerase chain reaction analysis of cultured cells demonstrated expression of procollagen 1-alpha1, DDR2, fibronectin, and vimentin mRNA. Fibroblast colonies consisting of EGFP(+) cells were observed in cultures of bone marrow cells from clonally engrafted mice, indicating an HSC origin of fibroblast colony-forming units. Culture of peripheral blood nucleated cells from clonally engrafted mice revealed EGFP(+) cells expressing collagen-I and DDR2, indicating that fibrocytes are also derived from HSCs. CONCLUSION: We conclude that a population of fibroblasts and their precursors are derived from HSCs.
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Ensayo de Unidades Formadoras de Colonias , Fibroblastos/metabolismo , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Trasplante Homólogo , Animales , Células Cultivadas , Células Clonales , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Receptores con Dominio Discoidina , Femenino , Fibroblastos/citología , Fibronectinas/genética , Fibronectinas/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Mitogénicos/genética , Receptores Mitogénicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vimentina/genética , Vimentina/metabolismoRESUMEN
We previously demonstrated the ability of hematopoietic stem cells (HSCs) to generate glomerular mesangial cells by trans-planting clonal populations of cells derived from a single enhanced green fluorescent protein (EGFP)-positive HSC into lethally irradiated mice. To define more precisely the hematopoietic differentiation pathway through which mesangial cells are derived, we studied the relationship between mesangial cell expression and individual hematopoietic lineages by means of a transplantation strategy. In a series of clonal HSC transplantation experiments, we generated 3 mice engrafted predominantly by granulocytes and macrophages (GMs) and 4 mice engrafted with B-cells or with B-cells and T-cells. When the kidneys of these mice were analyzed, the mice exhibiting high GM lineage engraftment revealed much higher levels of EGFP-positive mesangial cells than those with predominantly lymphocyte engraftment. Fluorescence in situ hybridization analysis of the kidneys from a male recipient of an EGFP-positive female donor excluded cell fusion as the cause for the observed differentiation. These results support the notion that glomerular mesangial cells share their origin with GMs.
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Diferenciación Celular/fisiología , Mesangio Glomerular/fisiología , Células Progenitoras Mieloides/fisiología , Animales , Linfocitos B/citología , Linfocitos B/fisiología , Linfocitos B/trasplante , Femenino , Mesangio Glomerular/citología , Granulocitos/citología , Granulocitos/fisiología , Trasplante de Células Madre Hematopoyéticas , Masculino , Ratones , Ratones Transgénicos , Monocitos/citología , Monocitos/fisiología , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/trasplante , Linfocitos T/citología , Linfocitos T/fisiología , Linfocitos T/trasplanteRESUMEN
Herein, we investigated the role of VEGF signaling in the earliest events in vasculogenesis and found that it exerts critical effects shortly after mesodermal cells form by gastrulation. We showed that VEGF treatment of embryos caused an increase in the population of newly gastrulated mesodermal (NGM) cells that express the transcription factor TAL1. This increase in TAL1-positive cells was attributed to VEGF induction of VEGF receptor-2 (Flk1)-positive NGM cells that would normally not have been induced due to the limited availability of VEGF in the NGM. Evidence that VEGF-mediated induction of NGM cells is relevant to the endothelial lineage is the finding that induced TAL1-positive cells in the NGM formed ectopic structures whose cells exhibited characteristics of endothelial cells, including the ability to integrate into the vascular network and express the QH1 antigen. Finally, we showed that VEGF-induced TAL1 expression in the NGM which resulted in the formation of ectopic structures was mediated by Flk1 but not Flt1 signaling. In summary, we have established that VEGF signaling is critical to allocation of NGM to the endothelial lineage.
Asunto(s)
Embrión no Mamífero/fisiología , Endotelio Vascular/embriología , Gástrula/fisiología , Mesodermo/fisiología , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Secuencia de Bases , Coturnix/embriología , Cartilla de ADN , Embrión no Mamífero/efectos de los fármacos , Gástrula/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Mesodermo/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We investigated the role of vascular endothelial (VE)-cadherin in blood vessel morphogenesis and established a temporal correlation linking the failure in vessel morphogenesis in VE-cadherin null embryos to a specific step in vasculogenesis. We showed that the sequence in which blood vessels failed followed the order in which they had formed (ie, those forming first--yolk sac, allantoic and endocardial vessels--were the first to display morphologic abnormalities). We next showed that in place of normal reticulated networks of blood vessels, clusters of platelet endothelial cell adhesion molecule-positive (PECAM+) cells formed within cultured allantois explants from VE-cadherin null embryos. Similarly, a function-blocking VE-cadherin antibody, BV13, caused PECAM+ cell clusters to form in cultured allantois explants from normal mice. Finally, we demonstrated that formation of PECAM+ cell clusters in response to BV13 was not due to a disruption in the formation of nascent vessels but was due to the actual disassembly of nascent vessels. Based on these findings, we conclude that the events of de novo blood vessel formation up to the point at which a vascular epithelium forms (ie, nascent vessels with lumens) are not dependent on VE-cadherin and that VE-cadherin, whose expression is up-regulated following vascular epithelialization, is required to prevent the disassembly of nascent blood vessels.
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Vasos Sanguíneos/embriología , Vasos Sanguíneos/fisiología , Cadherinas/genética , Cadherinas/metabolismo , Neovascularización Fisiológica/fisiología , Animales , Anticuerpos Monoclonales , Antígenos CD , Cadherinas/inmunología , Membrana Corioalantoides/embriología , Membrana Corioalantoides/fisiología , Endotelio/fisiología , Regulación del Desarrollo de la Expresión Génica , Mesodermo/fisiología , Ratones , Técnicas de Cultivo de Órganos , Saco Vitelino/embriología , Saco Vitelino/fisiologíaRESUMEN
Here we have investigated the role of sphingosine-1-phosphate (S1P) signaling in the process of vasculogenesis in the mouse embryo. At stages preceding the formation of blood vessels (7.5-8 dpc) in the embryo proper, yolk sac, and allantois, the S1P receptor S1P(2) is expressed in conjunction with S1P(1) and/or S1P(3). Additionally, sphingosine kinase-2 (SK2), an enzyme that catalyzes the formation of S1P, is expressed in these tissues throughout periods of vasculogenesis. Using the cultured mouse allantois explant model of blood vessel formation, we found that vasculogenesis was dependent on S1P signaling. We showed that S1P could replace the ability of serum to promote vasculogenesis in cultured allantois explants. Instead of small poorly reticulated clusters of rounded endothelial cells that formed under serum-free conditions, S1P promoted the formation of elongated endothelial cells that arranged into expansive branched networks of capillary-like vessels. These effects could not be reproduced by vascular endothelial growth factor or basic fibroblast growth factor administration. The ability of S1P to promote blood vessel formation was not due to effects on cell survival or on changes in numbers of endothelial cells (Flk1(+)/PECAM(+)), angioblasts (Flk1(+)/PECAM(-)), or undifferentiated mesodermal cells (Flk1(-)/PECAM(-)). The S1P effect on blood vessel formation was attributed to it promoting migratory activities of angioblasts and early endothelial cells required for the expansion of vascular networks. Together, our findings suggest that migratory events critical to the de novo formation of blood vessels are under the influence of S1P, possibly synthesized via the action of SK2, with signaling mediated by S1P receptors that include S1P(1), S1P(2), and S1P(3).
Asunto(s)
Movimiento Celular/fisiología , Lisofosfolípidos/metabolismo , Neovascularización Fisiológica/fisiología , Transducción de Señal/fisiología , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Alantoides/irrigación sanguínea , Animales , Ratones , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
BACKGROUND: Bone marrow (BM)-derived cells differentiate into a wide variety of cell types. BM contains a heterogeneous population of stem and progenitor cells including hematopoietic stem cells, marrow stromal cells, and perhaps other progenitor cells. To establish unequivocally the transdifferentiation capability of a hematopoietic cell to a nonhematopoietic cell (endothelial cells, neurons, and glial cells), it is imperative to demonstrate that a single cell or clone of that single cell (clonal analysis) differentiates into cells comprising vessels or other cells in the brain. METHODS: We generated mice that exhibited a high level of hematopoietic reconstitution from a single enhanced green fluorescent protein (EGFP) stem cell. To achieve this, we combined FACS sorting and cell culture to generate a population of cells derived from a single hematopoietic stem cell (Lin-, CD34-, c-kit+, and Sca-1+). Clonal populations of cells were then transplanted into lethally irradiated recipient mice. After 3-4 months of engraftment, some mice underwent middle cerebral artery (MCA) suture occlusion. EGFP immunocytochemistry and dual labeling was performed with cell-specific markers on tissue from various time points. RESULTS: In all transplanted mice, EGFP+ highly ramified cells were seen in the brain parenchyma. These cells stained with RCA120 lectin and had the characteristics of parenchymal microglial cells. In brains without infarction and in uninfarcted brain regions of mice that underwent MCA occlusion, there were many EGFP+ cells in a perivascular distribution, associated with both small and larger blood vessels. The cells were tightly apposed to the vessel wall and some had long processes that enveloped the endothelial cells. After MCA occlusion, there was an influx of EGFP expressing cells in the ischemic tissue that colocalized with the "neovascularization." These EGFP+ cells were wrapped around endothelial cells in an albuminal location and did not coexpress von Willebrand Factor or CD31. We detected rare dual-labeled EGFP and NeuN-expressing cells. We detected two staining patterns. The more frequent pattern was phagocytosis of NeuN cells by EGFP expressing cells. However, we also detected rarer cells where the EGFP and NeuN appeared to be colocalized by confocal microscopy. CONCLUSIONS: HSC differentiate into parenchymal microglial cells and perivascular cells in the brain. The numbers of these cells increase after cerebral ischemia. The HSC is therefore one source of parenchymal microglial cells and a source for perivascular cells. After a cerebral infarction, there are rare HSC-derived cells that stain with the neuronal marker, NeuN. However, the more common pattern appears to represent phagocytosis of damaged neurons by EGFP+ microglial cells.
Asunto(s)
Encéfalo/citología , Endotelio Vascular/citología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Microglía/citología , Animales , Antígenos/metabolismo , Bencimidazoles/metabolismo , Encéfalo/fisiología , Carbocianinas/metabolismo , Diferenciación Celular , Células Cultivadas , Células Endoteliales/metabolismo , Femenino , Citometría de Flujo/métodos , Lateralidad Funcional , Proteínas Fluorescentes Verdes , Trasplante de Células Madre Hematopoyéticas/métodos , Inmunohistoquímica/métodos , Infarto de la Arteria Cerebral Media/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal/métodos , Neuronas/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Lectinas de Plantas/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Quimera por Radiación , Factores de Tiempo , Factor de von Willebrand/inmunologíaRESUMEN
Cubilin and megalin are cell surface proteins that work cooperatively in many absorptive epithelia to mediate endocytosis of lipoproteins, vitamin carriers, and other proteins. Here we have investigated the coordinate expression of these receptors during mouse development. Our findings indicate that while there are sites where the receptors are co-expressed, there are other tissues where expression is not overlapping. Apical cubilin expression is pronounced in the extraembryonic visceral endoderm (VE) of 6-9.5 days postcoitum (dpc) embryos. By contrast, little megalin expression is evident in the VE at 6 dpc. However, megalin expression in the VE increases as development progresses (7.5-9.5 dpc), although it is not as uniformly distributed as cubilin. Punctate expression of megalin is also apparent in the region of the ectoplacental cone associated with decidual cells, whereas cubilin expression is not seen in association with the ectoplacenta. Strong expression of megalin is observed in the neural ectoderm, neural plate and neural tube (6-8.5 dpc), but cubilin expression is not apparent in any of these tissues. At 8.5 dpc, megalin is expressed in the developing endothelial cells of blood islands, whereas cubilin is absent from these cells. Finally, cubilin, but not megalin, is expressed by a subpopulation of cells dispersed within the 7.5 dpc embryonic endoderm and having a migratory morphology. In summary, the co-expression of cubilin and megalin in the VE is consistent with the two proteins functioning jointly in this tissue. However, the differential distribution pattern indicates that the proteins also function independent of one another. Furthermore, the finding of megalin expression in blood island endothelial cells and cubilin expression in embryonic endoderm highlight potential new developmental roles for these proteins.
Asunto(s)
Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/biosíntesis , Receptores de Superficie Celular/biosíntesis , Animales , Embrión de Mamíferos/química , Embrión de Mamíferos/embriología , Femenino , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/análisis , Ratones , Receptores de Superficie Celular/análisisRESUMEN
Morphometric methods were developed to characterize the geometry of vascular patterns in avian and murine embryos. By using these methods, we found that networks of blood vessels formed during vasculogenesis share similar geometric properties (i.e., mean blood vessel diameters and avascular space diameters) regardless of developmental stage, location, or species in which they form. We also found that endothelial cell density within a unit area of an embryonic vasculature could be used to accurately distinguish between a small diameter, capillary-like vascular network (low endothelial cell density) and a large diameter, presinusoidal network (high endothelial cell density). Furthermore, we show that endothelial cell size remains constant in small and large diameter vessels, indicating that increased endothelial cell size is not the basis for diversity in vessel diameter. These observations serve as a foundation for future studies seeking to evaluate the effects of agents or genetic mutations on aspects of vasculogenesis.
Asunto(s)
Vasos Sanguíneos/citología , Vasos Sanguíneos/embriología , Endotelio Vascular/citología , Endotelio Vascular/embriología , Alantoides/citología , Animales , Vasos Sanguíneos/ultraestructura , Recuento de Células , Tamaño de la Célula , Coturnix/embriología , Embrión de Mamíferos , Embrión no Mamífero , Endotelio Vascular/ultraestructura , Ratones/embriología , Microscopía Confocal , Morfogénesis , Técnicas de Cultivo de Órganos , Somitos , Factores de TiempoRESUMEN
Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPeta), participates in developmental vascularization. A mutant allele, CD148(DeltaCyGFP), was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions.