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1.
PLoS Pathog ; 20(1): e1011280, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38271464

RESUMEN

Subverting the host immune response to inhibit inflammation is a key virulence strategy of Yersinia pestis. The inflammatory cascade is tightly controlled via the sequential action of lipid and protein mediators of inflammation. Because delayed inflammation is essential for Y. pestis to cause lethal infection, defining the Y. pestis mechanisms to manipulate the inflammatory cascade is necessary to understand this pathogen's virulence. While previous studies have established that Y. pestis actively inhibits the expression of host proteins that mediate inflammation, there is currently a gap in our understanding of the inflammatory lipid mediator response during plague. Here we used the murine model to define the kinetics of the synthesis of leukotriene B4 (LTB4), a pro-inflammatory lipid chemoattractant and immune cell activator, within the lungs during pneumonic plague. Furthermore, we demonstrated that exogenous administration of LTB4 prior to infection limited bacterial proliferation, suggesting that the absence of LTB4 synthesis during plague contributes to Y. pestis immune evasion. Using primary leukocytes from mice and humans further revealed that Y. pestis actively inhibits the synthesis of LTB4. Finally, using Y. pestis mutants in the Ysc type 3 secretion system (T3SS) and Yersinia outer protein (Yop) effectors, we demonstrate that leukocytes recognize the T3SS to initiate the rapid synthesis of LTB4. However, several Yop effectors secreted through the T3SS effectively inhibit this host response. Together, these data demonstrate that Y. pestis actively inhibits the synthesis of the inflammatory lipid LTB4 contributing to the delay in the inflammatory cascade required for rapid recruitment of leukocytes to sites of infection.


Asunto(s)
Peste , Yersinia pestis , Humanos , Animales , Ratones , Yersinia pestis/metabolismo , Peste/microbiología , Sistemas de Secreción Tipo III/metabolismo , Leucotrieno B4/metabolismo , Leucocitos/metabolismo , Inflamación , Proteínas Bacterianas/metabolismo
2.
bioRxiv ; 2024 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-39229191

RESUMEN

OBJECTIVE: Diet-induced white adipose tissue inflammation is associated with insulin resistance and metabolic perturbations. Conversely, exercise (Exe) protects against the development of chronic inflammation and insulin resistance independent of changes in weight; however, the mechanisms remain largely unknown. We have recently shown that, through adrenergic stimulation of macrophages, exercise promotes resolution of acute peritoneal inflammation by enhancing the biosynthesis of specialized pro-resolving lipid mediators (SPMs). In this study, we sought to determine if exercise stimulates pro-resolving pathways in adipose tissue and whether this response is modified by diet. Specifically, we hypothesized that high fat diet feeding disrupts exercise-stimulated resolution by inhibiting adrenergic signaling, priming the development of chronic inflammation in adipose tissue (AT). APPROACH AND RESULTS: To explore the dietary dependence of the pro-resolving effects of Exe, mice were fed either a control or high-fat diet (HFD) for 2 weeks prior to, and throughout, a 4 wk period of daily treadmill running. Glucose handling, body weight and composition, and exercise performance were evaluated at the end of the feeding and exercise interventions. Likewise, catecholamines and their biosynthetic enzymes were measured along with AT SPM biosynthesis and macrophage phenotype and abundance. When compared with sedentary controls (Sed), macrophages isolated from mice exposed to 4 wk of exercise display elevated expression of the SPM biosynthetic enzyme Alox15, while whole AT SPM levels and anti-inflammatory CD301+ M2 macrophages increased. These changes were dependent upon diet as 6 wk of feeding with HFD abrogated the pro-resolving effect of exercise when compared with control diet-fed animals. Interestingly, exercise-induced epinephrine production was inhibited by HFD, which diminished expression of the epinephrine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT) in adrenal glands. CONCLUSION: Taken together, these results suggest that a diet high in fat diminishes the pro-resolving effects of exercise in adipose tissue via decreasing the biosynthesis of catecholamines.

3.
Am J Physiol Cell Physiol ; 304(4): C289-98, 2013 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-23220114

RESUMEN

The objective of this study was to develop a novel in vitro model for smooth muscle cell (SMC) differentiation from human embryonic stem cell-derived mesenchymal cells (hES-MCs). We found that hES-MCs were differentiated to SMCs by transforming growth factor-ß (TGF-ß) in a dose- and time-dependent manner as demonstrated by the expression of SMC-specific genes smooth muscle α-actin, calponin, and smooth muscle myosin heavy chain. Under normal growth conditions, however, the differentiation capacity of hES-MCs was very limited. hES-MC-derived SMCs had an elongated and spindle-shaped morphology and contracted in response to the induction of carbachol and KCl. KCl-induced calcium transient was also evident in these cells. Compared with the parental cells, TGF-ß-treated hES-MCs sustained the endothelial tube formation for a longer time due to the sustained SMC phenotype. Mechanistically, TGF-ß-induced differentiation was both Smad- and serum response factor/myocardin dependent. TGF-ß regulated myocardin expression via multiple signaling pathways including Smad2/3, p38 MAPK, and PI3K. Importantly, we found that a low level of myocardin was present in mesoderm prior to SMC lineage determination, and a high level of myocardin was not induced until the differentiation process was initiated. Taken together, our study characterized a novel SMC differentiation model that can be used for studying human SMC differentiation from mesoderm during vascular development.


Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/fisiología , Células Madre Mesenquimatosas/fisiología , Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Señalización del Calcio , Técnicas de Cultivo de Célula , Forma de la Célula , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/fisiología , Expresión Génica , Humanos , Contracción Muscular , Desarrollo de Músculos , Miocitos del Músculo Liso/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Factor de Respuesta Sérica/metabolismo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Activación Transcripcional , Factor de Crecimiento Transformador beta/fisiología
4.
Biotechnol Bioeng ; 109(1): 274-83, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21837664

RESUMEN

Human mesenchymal stem cells (hMSC) have proven beneficial in the repair and preservation of infarcted myocardium. Unfortunately, MSCs represent a small portion of the bone marrow and require ex vivo expansion. To further advance the clinical usefulness of cellular cardiomyoplasty, derivation of "MSC-like" cells that can be made available "off-the-shelf" are desirable. Recently, human embryonic stem cell-derived mesenchymal cells (hESC-MC) were described. We investigated the efficacy of hESC-MC for cardiac repair after myocardial infarction (MI) compared to hMSC. Because of increased efficacy of cell delivery, cells were embedded into collagen patches and delivered to infarcted myocardium. Culture of hMSC and hESC-MCs in collagen patches did not induce differentiation or significant loss in viability. Transplantation of hMSC and hES-MC patches onto infarcted myocardium of athymic nude rats prevented adverse changes in infarct wall thickness and fractional area change compared to a non-viable patch control. Hemodynamic assessment showed that hMSCs and hES-MC patch application improved end diastolic pressure equivalently. There were no changes in systolic function. hES-MC and hMSC construct application enhanced neovessel formation compared to a non-viable control, and each cell type had similar efficacy in stimulating endothelial cell growth in vitro. In summary, the use of hES-MC provides similar efficacy for cellular cardiomyoplasty as compared to hMSC and may be considered a suitable alternative for cell therapy.


Asunto(s)
Células Madre Embrionarias/fisiología , Infarto del Miocardio/terapia , Ingeniería de Tejidos/métodos , Animales , Presión Sanguínea/fisiología , Diferenciación Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Humanos , Células Madre Mesenquimatosas/fisiología , Ratas , Resultado del Tratamiento
5.
Mol Metab ; 66: 101637, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36400404

RESUMEN

OBJECTIVE: Physical activity has been shown to reduce the risk of CVD mortality in large-cohort longitudinal studies; however, the mechanisms underpinning the beneficial effects of exercise remain incompletely understood. Emerging data suggest that the risk reducing effect of exercise extends beyond changes in traditional CVD risk factors alone and involves alterations in immunity and reductions in inflammatory mediator production. Our study aimed to determine whether exercise-enhanced production of proresolving lipid mediators contribute to alterations in macrophage intermediary metabolism, which may contribute to the anti-inflammatory effects of exercise. METHODS: Changes in lipid mediators and macrophage metabolism were assessed in C57Bl/6 mice following 4 weeks of voluntary exercise training. To investigate whether exercise-stimulated upregulation of specialized proresolving lipid mediators (SPMs) was sufficient to enhance mitochondrial respiration, both macrophages from control mice and human donors were incubated in vitro with SPMs and mitochondrial respiratory parameters were measured using extracellular flux analysis. Compound-C, an ATP-competitive inhibitor of AMPK kinase activity, was used to investigate the role of AMPK activity in SPM-induced mitochondrial metabolism. To assess the in vivo contribution of 5-lipoxygenase in AMPK activation and exercise-induced mitochondrial metabolism in macrophages, Alox5-/- mice were also subjected to exercise training. RESULTS: Four weeks of exercise training enhanced proresolving lipid mediator production, while also stimulating the catabolism of inflammatory lipid mediators (e.g., leukotrienes and prostaglandins). This shift in lipid mediator balance following exercise was associated with increased macrophage mitochondrial metabolism. We also find that treating human and murine macrophages in vitro with proresolving lipid mediators enhances mitochondrial respiratory parameters. The proresolving lipid mediators RvD1, RvE1, and MaR1, but not RvD2, stimulated mitochondrial respiration through an AMPK-dependent signaling mechanism. Additionally, in a subset of macrophages, exercise-induced mitochondrial activity in vivo was dependent upon 5-lipoxygenase activity. CONCLUSION: Collectively, these results suggest that exercise stimulates proresolving lipid mediator biosynthesis and mitochondrial metabolism in macrophages via AMPK, which might contribute to the anti-inflammatory and CVD risk reducing effect of exercise.


Asunto(s)
Proteínas Quinasas Activadas por AMP , Ejercicio Físico , Macrófagos , Animales , Humanos , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Araquidonato 5-Lipooxigenasa/farmacología , Enfermedades Cardiovasculares/metabolismo , Macrófagos/metabolismo , Fosforilación , Ejercicio Físico/fisiología , Respiración de la Célula/fisiología , Mitocondrias/metabolismo , Mitocondrias/fisiología , Inflamación/metabolismo
6.
Stem Cells Dev ; 30(12): 632-640, 2021 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-34029164

RESUMEN

Homozygous familial hypercholesterolemia (hoFH) is a rare disorder caused primarily by pathological mutations in the low-density lipoprotein receptor (LDLR), which disrupts LDL-cholesterol (LDL-C) metabolism homeostasis. hoFH patients are at extremely high risk for cardiovascular disease and are resistant to standard therapies. LDLR knockout animals and in vitro cell models overexpressing different mutations have proved useful, but may not fully recapitulate human LDLR mutation biology. We and others have generated induced pluripotent stem cells (iPSC) from hoFH patient's fibroblasts and T cells and demonstrated their ability to recapitulate hoFH biology. In this study, we present the generation and characterization of a cohort of seven hoFH-iPSC lines derived from peripheral blood mononuclear cells (PBMC) collected from four homozygous and three compound heterozygous patients. The hoFH-iPSC cohort demonstrated a wide range of LDLR expression and LDL-C internalization in response to rosuvastatin that correlated with the predicted pathogenicity of the mutation. We were able to confirm that hoFH-iPSC cohort were pluripotent by differentiation toward all three germ layers and specifically to hepatocyte-like cells (HLC), the cell with primary LDL-C metabolic regulatory control, by expression of hepatocyte markers. hoFH patient PBMC-derived iPSC recapitulate the LDLR dysfunction of their specific mutation. They were capable of differentiating to HLC and could be useful for early developmental studies, pharmacology/toxicology, and potentially autologous cell therapy.


Asunto(s)
Hiperlipoproteinemia Tipo II , Células Madre Pluripotentes Inducidas , LDL-Colesterol/genética , LDL-Colesterol/metabolismo , Homocigoto , Humanos , Hiperlipoproteinemia Tipo II/genética , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Mutación/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo
7.
Dis Model Mech ; 13(4)2020 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-32005714

RESUMEN

Low-density lipoprotein (LDL) receptor (LDLR) mutations are the primary cause of familial hypercholesterolemia (FH). Class II LDLR mutations result in a misfolded LDLR retained in the endoplasmic reticulum (ER). We have developed a model of FH class II and CRISPR-corrected induced pluripotent stem cells (iPSC) capable of replicating mutant and repaired LDLR functions. We show here that iPSC and derived hepatocyte-like cells (HLC) replicate misfolded LDLR accumulation and restoration of LDLR function in CRISPR-corrected cells. It was reported that model cells overexpressing class II LDLR mutants result in endoplasmic reticulum (ER) accumulation of immature LDLR and activation of the unfolded protein response (UPR). We show here that statins induce a similar accumulation of immature LDLR that is resolved with class II correction. We also demonstrate that, although capable of UPR induction with tunicamycin treatment, unlike overexpression models, statin-treated class II iPSC and derived HLC do not induce the common UPR markers Grp78 (also known as HSPA5) or spliced XBP1 [XBP1 (S)]. Because statins are reported to inhibit UPR, we utilized lipoprotein-deficient serum (LPDS) medium, but still did not detect UPR induction at the Grp78 and XBP1 (S) levels. Our study demonstrates the recapitulation of mutant and corrected class II LDLR function and suggests that overexpression models may not accurately predict statin-mediated class II protein biology.


Asunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Hiperlipoproteinemia Tipo II/tratamiento farmacológico , Hiperlipoproteinemia Tipo II/metabolismo , Receptores de LDL/metabolismo , Calnexina/metabolismo , Endocitosis/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Modelos Biológicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de LDL/genética , Rosuvastatina Cálcica/farmacología , Rosuvastatina Cálcica/uso terapéutico , Respuesta de Proteína Desplegada/efectos de los fármacos
8.
Stem Cells ; 26(11): 2768-76, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18719225

RESUMEN

Human embryonic stem cells (hESCs) have recently demonstrated the potential for differentiation into germ-like cells in vitro. This provides a novel model for understanding human germ cell development and human infertility. Mouse embryonic fibroblast (MEF) feeders and basic fibroblast growth factor (bFGF) are two sources of signaling that are essential for primary culture of germ cells, yet their role has not been examined in the derivation of germ-like cells from hESCs. Here protein and gene expression demonstrated that both MEF feeders and bFGF can significantly enrich germ cell differentiation from hESCs. Under enriched differentiation conditions, flow cytometry analysis proved 69% of cells to be positive for DDX4 and POU5F1 protein expression, consistent with the germ cell lineage. Importantly, removal of bFGF from feeder-free cultures resulted in a 50% decrease in POU5F1- and DDX4-positive cells. Quantitative reverse transcription-polymerase chain reaction analysis established that bFGF signaling resulted in an upregulation of genes involved in germ cell differentiation with or without feeders; however, feeder conditions caused significant upregulation of premigratory/migratory (Ifitm3, DAZL, NANOG, and POU5F1) and postmigratory (PIWIL2, PUM2) genes, along with the meiotic markers SYCP3 and MLH1. After further differentiation, >90% of cells expressed the meiotic proteins SYCP3 and MLH1. This is the first demonstration that signaling from MEF feeders and bFGF can induce a highly enriched population of germ-like cells derived from hESCs, thus providing a critically needed model for further investigation of human germ cell development and signaling. Disclosure of potential conflicts of interest is found at the end of this article.


Asunto(s)
Células Madre Embrionarias/citología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Células Germinativas/citología , Animales , Antígenos de Diferenciación/metabolismo , Diferenciación Celular , Técnicas de Cocultivo , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/fisiología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/citología , Humanos , Ratones , Transducción de Señal
9.
Differentiation ; 76(5): 454-64, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18177420

RESUMEN

Derivation of human neural progenitors (hNP) from human embryonic stem (hES) cells in culture has been reported with the use of feeder cells or conditioned media. This introduces undefined components into the system, limiting the ability to precisely investigate the requirement for factors that control the process. Also, the use of feeder cells of non-human origin introduces the potential for zoonotic transmission, limiting its clinical usefulness. Here we report a feeder-free system to produce hNP from hES cells and test the effects of various media components involved in the process. Five protocols using defined media components were compared for efficiency of hNP generation. Based on this analysis, we discuss the role of basic fibroblast growth factor (FGF2), N2 supplement, non-essential amino acids (NEAA), and knock-out serum replacement (KSR) on the process of hNP generation. All protocols led to down-regulation of Oct4/POU5F1 expression (from 90.5% to <3%), and up-regulation of neural progenitor markers to varying degrees. Media with N2 but not KSR and NEAA produced cultures with significantly higher (p<0.05) expression of the neural progenitor marker Musashi 1 (MSI1). Approximately 89% of these cells were Nestin (NES)+ after 3 weeks, but they did not proliferate. In contrast, differentiation media supplemented with KSR and NEAA produced fewer NES+ (75%) cells, but these cells were proliferative, and by five passages the culture consisted of >97% NES+ cells. This suggests that KSR and NEAA supplements did not enhance early differentiation but did promote proliferating of hNP cell cultures. This resulted in an efficient, robust, repeatable differentiation system suitable for generating large populations of hNP cells. This will facilitate further study of molecular and biochemical mechanisms in early human neural differentiation and potentially produce uniform neuronal cells for therapeutic uses without concern of zoonotic transmission from feeder layers.


Asunto(s)
Astrocitos/citología , Técnicas de Cultivo de Célula/métodos , Medios de Cultivo/farmacología , Células Madre Embrionarias/citología , Neuronas/citología , Oligodendroglía/citología , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Astrocitos/metabolismo , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Medios de Cultivo/química , Medios de Cultivo Condicionados/farmacología , Medio de Cultivo Libre de Suero/farmacología , Células Madre Embrionarias/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Laminina , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Oligodendroglía/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reproducibilidad de los Resultados
10.
Tissue Eng Part B Rev ; 24(4): 289-299, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-28316259

RESUMEN

A major challenge in tissue engineering is the generation of sufficient volumes of viable tissue for organ transplant. The development of a stable, mature vasculature is required to sustain the metabolic and functional activities of engineered tissues. Adipose stromal vascular fraction (SVF) cells are an easily accessible, heterogeneous cell system comprised of endothelial cells, macrophages, pericytes, and various stem cell populations. Collectively, SVF has been shown to spontaneously form vessel-like networks in vitro and robust, patent, and functional vasculatures in vivo. Capitalizing on this ability, we and others have demonstrated adipose SVF's utility in generating and augmenting engineered liver, cardiac, and vascular tissues, to name a few. This review highlights the scientific origins of SVF, the use of SVF as a clinically relevant vascular source, various SVF constituents and their roles, and practical considerations associated with isolating SVF for various tissue engineering applications.


Asunto(s)
Tejido Adiposo , Separación Celular/métodos , Neovascularización Fisiológica , Células Madre , Ingeniería de Tejidos/métodos , Tejido Adiposo/irrigación sanguínea , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Animales , Humanos , Células Madre/citología , Células Madre/metabolismo
11.
Hepatol Commun ; 1(9): 886-898, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29130076

RESUMEN

Familial hypercholesterolemia (FH) is a hereditary disease primarily due to mutations in the low-density lipoprotein receptor (LDLR) that lead to elevated cholesterol and premature development of cardiovascular disease. Homozygous FH patients (HoFH) with two dysfunctional LDLR alleles are not as successfully treated with standard hypercholesterol therapies, and more aggressive therapeutic approaches to control cholesterol levels must be considered. Liver transplant can resolve HoFH, and hepatocyte transplantation has shown promising results in animals and humans. However, demand for donated livers and high-quality hepatocytes overwhelm the supply. Human pluripotent stem cells can differentiate to hepatocyte-like cells (HLCs) with the potential for experimental and clinical use. To be of future clinical use as autologous cells, LDLR genetic mutations in derived FH-HLCs need to be corrected. Genome editing technology clustered-regularly-interspaced-short-palindromic-repeats/CRISPR-associated 9 (CRISPR/Cas9) can repair pathologic genetic mutations in human induced pluripotent stem cells. CONCLUSION: We used CRISPR/Cas9 genome editing to permanently correct a 3-base pair homozygous deletion in LDLR exon 4 of patient-derived HoFH induced pluripotent stem cells. The genetic correction restored LDLR-mediated endocytosis in FH-HLCs and demonstrates the proof-of-principle that CRISPR-mediated genetic modification can be successfully used to normalize HoFH cholesterol metabolism deficiency at the cellular level.

12.
Sci Rep ; 7(1): 16652, 2017 11 30.
Artículo en Inglés | MEDLINE | ID: mdl-29192272

RESUMEN

We report here that in rat and human neuroprogenitor cells as well as rat embryonic cortical neurons Zika virus (ZIKV) infection leads to ribosomal stress that is characterized by structural disruption of the nucleolus. The anti-nucleolar effects were most pronounced in postmitotic neurons. Moreover, in the latter system, nucleolar presence of ZIKV capsid protein (ZIKV-C) was associated with ribosomal stress and apoptosis. Deletion of 22 C-terminal residues of ZIKV-C prevented nucleolar localization, ribosomal stress and apoptosis. Consistent with a casual relationship between ZIKV-C-induced ribosomal stress and apoptosis, ZIKV-C-overexpressing neurons were protected by loss-of-function manipulations targeting the ribosomal stress effector Tp53 or knockdown of the ribosomal stress mediator RPL11. Finally, capsid protein of Dengue virus, but not West Nile virus, induced ribosomal stress and apoptosis. Thus, anti-nucleolar and pro-apoptotic effects of protein C are flavivirus-species specific. In the case of ZIKV, capsid protein-mediated ribosomal stress may contribute to neuronal death, neurodevelopmental disruption and microcephaly.


Asunto(s)
Apoptosis , Proteínas de la Cápside/metabolismo , Neuronas/metabolismo , Ribosomas/metabolismo , Estrés Fisiológico , Proteína p53 Supresora de Tumor/metabolismo , Infección por el Virus Zika/metabolismo , Virus Zika/fisiología , Animales , Proteínas de la Cápside/genética , Nucléolo Celular/metabolismo , Células Cultivadas , Femenino , Expresión Génica , Interacciones Huésped-Patógeno , Neuronas/virología , Transporte de Proteínas , Ratas , Infección por el Virus Zika/virología
13.
Circ Res ; 95(8): 773-9, 2004 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-15388638

RESUMEN

Atherosclerosis is an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions including oscillatory shear stress (OS). OS exposure induces endothelial expression of bone morphogenic protein 4 (BMP4), which in turn may activate intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesion. OS is also known to induce monocyte adhesion by producing reactive oxygen species (ROS) from reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, raising the possibility that BMP4 may stimulate the inflammatory response by ROS-dependent mechanisms. Here we show that ROS scavengers blocked ICAM-1 expression and monocyte adhesion induced by BMP4 or OS in endothelial cells (ECs). Similar to OS, BMP4 stimulated H2O2 and O2- production in ECs. Next, we used ECs obtained from p47phox-/- mice (MAE-p47-/-), which do not produce ROS in response to OS, to determine the role of NADPH oxidases. Similar to OS, BMP4 failed to induce monocyte adhesion in MAE-p47-/-, but it was restored when the cells were transfected with p47phox plasmid. Moreover, OS-induced O2- production was blocked by noggin (a BMP antagonist), suggesting a role for BMP. Furthermore, OS increased gp91phox (nox2) and nox1 mRNA levels while decreasing nox4. In contrast, BMP4 induced nox1 mRNA expression, whereas nox2 and nox4 were decreased or not affected, respectively. Also, OS-induced monocyte adhesion was blocked by knocking down nox1 with the small interfering RNA (siRNA). Finally, BMP4 siRNA inhibited OS-induced ROS production and monocyte adhesion. Together, these results suggest that BMP4 produced in ECs by OS stimulates ROS release from the nox1-dependent NADPH oxidase leading to inflammation, a critical early atherogenic step.


Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Regulación de la Expresión Génica , Monocitos/citología , NADH NADPH Oxidorreductasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Mecánico , Animales , Antioxidantes/farmacología , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/biosíntesis , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/farmacología , Adhesión Celular , Células Cultivadas/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Ratones , Ratones Endogámicos C57BL , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , NADPH Oxidasas , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes/farmacología , Reología , Superóxidos/metabolismo , Transfección
14.
Reprod Fertil Dev ; 18(8): 839-46, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17147932

RESUMEN

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.


Asunto(s)
Células Madre Embrionarias/fisiología , Animales , Diferenciación Celular/fisiología , Humanos , Cariotipificación , Macaca mulatta , Ratones , Neuronas/fisiología , Fenotipo , Transducción de Señal/fisiología
15.
PLoS One ; 11(3): e0151402, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26963616

RESUMEN

Human adipose-derived stromal vascular fraction (hSVF) cells are an easily accessible, heterogeneous cell system that can spontaneously self-assemble into functional microvasculatures in vivo. However, the mechanisms underlying vascular self-assembly and maturation are poorly understood, therefore we utilized an in vitro model to identify potential in vivo regulatory mechanisms. We utilized passage one (P1) hSVF because of the rapid UEA1+ endothelium (EC) loss at even P2 culture. We exposed hSVF cells to a battery of angiogenesis inhibitors and found that the pan-Wnt inhibitor IWP2 produced the most significant hSVF-EC networking decrease (~25%). To determine which Wnt isoform(s) and receptor(s) may be involved, hSVF was screened by PCR for isoforms associated with angiogenesis, with only WNT5A and its receptor, FZD4, being expressed for all time points observed. Immunocytochemistry confirmed Wnt5a protein expression by hSVF. To see if Wnt5a alone could restore IWP2-induced EC network inhibition, recombinant human Wnt5a (0-150 ng/ml) was added to IWP2-treated cultures. The addition of rhWnt5a significantly increased EC network area and significantly decreased the ratio of total EC network length to EC network area compared to untreated controls. To determine if Wnt5a mediates in vivo microvascular self-assembly, 3D hSVF constructs containing an IgG isotype control, anti-Wnt5a neutralizing antibody or rhWnt5a were implanted subcutaneously for 2w in immune compromised mice. Compared to IgG controls, anti-Wnt5a treatment significantly reduced vessel length density by ~41%, while rhWnt5a significantly increased vessel length density by ~62%. However, anti-Wnt5a or rhWnt5a did not significantly affect the density of segments and nodes, both of which measure vascular complexity. Taken together, this data demonstrates that endogenous Wnt5a produced by hSVF plays a regulatory role in microvascular self-assembly in vivo. These findings also suggest that manipulating Wnt signaling could enhance control of hSVF vascularization in tissue engineering applications.


Asunto(s)
Adipocitos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Microvasos/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Proto-Oncogénicas/farmacología , Proteínas Wnt/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Adipocitos/citología , Adipocitos/metabolismo , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Ratones , Microvasos/metabolismo , Neovascularización Fisiológica/fisiología , Vía de Señalización Wnt/fisiología , Proteína Wnt-5a
16.
Cell Transplant ; 24(10): 2029-39, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25397993

RESUMEN

Adipose-derived stromal vascular fraction (SVF) cells have been shown to self-associate to form vascular structures under both in vitro and in vivo conditions. The angiogenic (new vessels from existing vessels) and vasculogenic (new vessels through self-assembly) potential of the SVF cell population may provide a cell source for directly treating (i.e., point of care without further cell isolation) ischemic tissues. However the correct dosage of adipose SVF cells required to achieve a functional vasculature has not been established. Accordingly, in vitro and in vivo dose response assays were performed evaluating the SVF cell vasculogenic potential. Serial dilutions of freshly isolated rat adipose SVF cells were plated on growth factor reduced Matrigel and vasculogenesis, assessed as cellular tube-like network assembly, was quantified after 3 days of culture. This in vitro vasculogenesis assay indicated that rat SVF cells reached maximum network length at a concentration of 2.5 × 10(5) cells/ml and network maintained at the higher concentrations tested. The same concentrations of rat and human SVF cells were used to evaluate vasculogenesis in vivo. SVF cells were incorporated into collagen gels and subcutaneously implanted into Rag1 immunodeficient mice. The 3D confocal images of harvested constructs were evaluated to quantify dose dependency of SVF cell vasculogenesis potential. Rat- and human-derived SVF cells yielded a maximum vasculogenic potential at 1 × 10(6) and 4 × 10(6) cells/ml, respectively. No adverse reactions (e.g., toxicity, necrosis, tumor formation) were observed at any concentration tested. In conclusion, the vasculogenic potential of adipose-derived SVF cell populations is dose dependent.


Asunto(s)
Adipocitos/citología , Tejido Adiposo/citología , Diferenciación Celular/fisiología , Neovascularización Patológica/patología , Células del Estroma/citología , Adiposidad/fisiología , Animales , Células Cultivadas , Humanos , Ratones , Neovascularización Patológica/metabolismo , Ratas
17.
Sci Rep ; 5: 13231, 2015 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-26307169

RESUMEN

Acquiring sufficient amounts of high-quality cells remains an impediment to cell-based therapies. Induced pluripotent stem cells (iPSC) may be an unparalleled source, but autologous iPSC likely retain deficiencies requiring correction. We present a strategy for restoring physiological function in genetically deficient iPSC utilizing the low-density lipoprotein receptor (LDLR) deficiency Familial Hypercholesterolemia (FH) as our model. FH fibroblasts were reprogrammed into iPSC using synthetic modified mRNA. FH-iPSC exhibited pluripotency and differentiated toward a hepatic lineage. To restore LDLR endocytosis, FH-iPSC were transfected with a 31 kb plasmid (pEHZ-LDLR-LDLR) containing a wild-type LDLR (FH-iPSC-LDLR) controlled by 10 kb of upstream genomic DNA as well as Epstein-Barr sequences (EBNA1 and oriP) for episomal retention and replication. After six months of selective culture, pEHZ-LDLR-LDLR was recovered from FH-iPSC-LDLR and transfected into Ldlr-deficient CHO-a7 cells, which then exhibited feedback-controlled LDLR-mediated endocytosis. To quantify endocytosis, FH-iPSC ± LDLR were differentiated into mesenchymal cells (MC), pretreated with excess free sterols, Lovastatin, or ethanol (control), and exposed to DiI-LDL. FH-MC-LDLR demonstrated a physiological response, with virtually no DiI-LDL internalization with excess sterols and an ~2-fold increase in DiI-LDL internalization by Lovastatin compared to FH-MC. These findings demonstrate the feasibility of functionalizing genetically deficient iPSC using episomal plasmids to deliver physiologically responsive transgenes.


Asunto(s)
Endocitosis/genética , Hiperlipoproteinemia Tipo II/genética , Células Madre Pluripotentes Inducidas/patología , Células Madre Pluripotentes Inducidas/fisiología , Plásmidos/genética , Receptores de LDL/genética , Diferenciación Celular/genética , Células Cultivadas , Mejoramiento Genético/métodos , Humanos , Plásmidos/administración & dosificación , Recuperación de la Función
18.
Tissue Eng Part A ; 20(1-2): 365-77, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23952622

RESUMEN

The in vivo osteogenesis potential of mesenchymal-like cells derived from human embryonic stem cells (hESC-MCs) was evaluated in vivo by implantation on collagen/hydroxyapatite scaffolds into calvarial defects in immunodeficient mice. This study is novel because no osteogenic or chondrogenic differentiation protocols were applied to the cells prior to implantation. After 6 weeks, X-ray, microCT, and histological analysis showed that the hESC-MCs had consistently formed a highly vascularized new bone that bridged the bone defect and seamlessly integrated with host bone. The implanted hESC-MCs differentiated in situ to functional hypertrophic chondrocytes, osteoblasts, and osteocytes forming new bone tissue via an endochondral ossification pathway. Evidence for the direct participation of the human cells in bone morphogenesis was verified by two separate assays: with Alu and by human mitochondrial antigen positive staining in conjunction with co-localized expression of human bone sialoprotein in histologically verified regions of new bone. The large volume of new bone in a calvarial defect and the direct participation of the hESC-MCs far exceeds that of previous studies and that of the control adult hMSCs. This study represents a key step forward for bone tissue engineering because of the large volume, vascularity, and reproducibility of new bone formation and the discovery that it is advantageous to not over-commit these progenitor cells to a particular lineage prior to implantation. The hESC-MCs were able to recapitulate the mesenchymal developmental pathway and were able to repair the bone defect semi-autonomously without preimplantation differentiation to osteo- or chondroprogenitors.


Asunto(s)
Regeneración Ósea/fisiología , Células Madre Embrionarias/citología , Células Madre Mesenquimatosas/citología , Adulto , Animales , Regeneración Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Colágeno/farmacología , Durapatita/farmacología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Morfogénesis/efectos de los fármacos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Fenotipo , Implantación de Prótesis , Regeneración/efectos de los fármacos , Cráneo/efectos de los fármacos , Cráneo/patología , Trasplante de Células Madre , Andamios del Tejido/química
19.
Tissue Eng Part A ; 19(1-2): 211-23, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22971005

RESUMEN

The microvasculature is principally composed of two cell types: endothelium and mural support cells. Multiple sources are available for human endothelial cells (ECs) but sources for human microvascular mural cells (MCs) are limited. We derived multipotent mesenchymal progenitor cells from human embryonic stem cells (hES-MC) that can function as an MC and stabilize human EC networks in three-dimensional (3D) collagen-fibronectin culture by paracrine mechanisms. Here, we have investigated the basis for hES-MC-mediated stabilization and identified the pleiotropic growth factor hepatocyte growth factor/scatter factor (HGF/SF) as a putative hES-MC-derived regulator of EC network stabilization in 3D in vitro culture. Pharmacological inhibition of the HGF receptor (Met) (1 µm SU11274) inhibits EC network formation in the presence of hES-MC. hES-MC produce and release HGF while human umbilical vein endothelial cells (HUVEC) do not. When HUVEC are cultured alone the networks collapse, but in the presence of recombinant human HGF or conditioned media from human HGF-transduced cells significantly more networks persist. In addition, HUVEC transduced to constitutively express human HGF also form stable networks by autocrine mechanisms. By enzyme-linked immunosorbent assay, the coculture media were enriched in both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2), but at significantly different levels (Ang1=159±15 pg/mL vs. Ang2=30,867±2685 pg/mL) contributed by hES-MC and HUVEC, respectively. Although the coculture cells formed stabile network architectures, their morphology suggests the assembly of an immature plexus. When HUVEC and hES-MC were implanted subcutaneously in immune compromised Rag1 mice, hES-MC increased their contact with HUVEC along the axis of the vessel. This data suggests that HUVEC and hES-MC form an immature plexus mediated in part by HGF and angiopoietins that is capable of maturation under the correct environmental conditions (e.g., in vivo). Therefore, hES-MC can function as microvascular MCs and may be a useful cell source for testing EC-MC interactions.


Asunto(s)
Proteínas Angiogénicas/metabolismo , Vasos Sanguíneos/citología , Vasos Sanguíneos/crecimiento & desarrollo , Células Madre Embrionarias/citología , Células Endoteliales/citología , Células Madre Mesenquimatosas/citología , Venas Umbilicales/citología , Animales , Técnicas de Cultivo Celular por Lotes/métodos , Comunicación Celular/fisiología , Células Cultivadas , Células Madre Embrionarias/metabolismo , Células Endoteliales/metabolismo , Factores de Crecimiento Endotelial/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Venas Umbilicales/metabolismo
20.
Tissue Eng Part A ; 17(11-12): 1537-48, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21284534

RESUMEN

Microvascular mural or perivascular cells are required for the stabilization and maturation of the remodeling vasculature. However, much less is known about their biology and function compared to large vessel smooth muscle cells. We have developed lines of multipotent mesenchymal cells from human embryonic stem cells (hES-MC); we hypothesize that these can function as perivascular mural cells. Here we show that the derived cells do not form teratomas in SCID mice and independently derived lines show similar patterns of gene expression by microarray analysis. When exposed to platelet-derived growth factor-BB, the platelet-derived growth factor receptor ß is activated and hES-MC migrate in response to a gradient. We also show that in a serum-free medium, transforming growth factor ß1 (TGFß1) induces robust expression of multiple contractile proteins (α smooth muscle actin, smooth muscle myosin heavy chain, smooth muscle 22α, and calponin). TGFß1 signaling is mediated through the TGFßR1/Alk5 pathway as demonstrated by inhibition of α smooth muscle actin expression by treatment of the Alk5-specific inhibitor SB525334 and stable retroviral expression of the Alk5 dominant negative (K232R). Coculture of human umbilical vein endothelial cell (HUVEC) with hES-MC maintains network integrity compared to HUVEC alone in three-dimensional collagen I-fibronectin by paracrine signaling. Using high-resolution laser confocal microscopy, we show that hES-MC also make direct contact with HUVEC. This demonstrates that hESC-derived mesenchymal cells possess the molecular machinery expected in a perivascular progenitor cells and can play a functional role in stabilizing EC networks in in vitro three-dimensional culture.


Asunto(s)
Células Madre Embrionarias/citología , Células Madre Mesenquimatosas/citología , Microvasos/citología , Actinas/metabolismo , Animales , Becaplermina , Línea Celular , Colágeno/farmacología , Proteínas Contráctiles/metabolismo , Medio de Cultivo Libre de Suero/farmacología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Fibronectinas/farmacología , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-sis , Ratas , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/efectos de los fármacos , Teratoma/patología , Factor de Crecimiento Transformador beta1/farmacología , Venas Umbilicales/citología
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