RESUMEN
All-trans-retinoic Acid (atRA) is the principal active metabolite of Vitamin A, essential for various biological processes. The activities of atRA are mediated by nuclear RA receptors (RARs) to alter gene expression (canonical activities) or by cellular retinoic acid binding protein 1 (CRABP1) to rapidly (minutes) modulate cytosolic kinase signaling, including calcium calmodulin-activated kinase 2 (CaMKII) (non-canonical activities). Clinically, atRA-like compounds have been extensively studied for therapeutic applications; however, RAR-mediated toxicity severely hindered the progress. It is highly desirable to identify CRABP1-binding ligands that lack RAR activity. Studies of CRABP1 knockout (CKO) mice revealed CRABP1 to be a new therapeutic target, especially for motor neuron (MN) degenerative diseases where CaMKII signaling in MN is critical. This study reports a P19-MN differentiation system, enabling studies of CRABP1 ligands in various stages of MN differentiation, and identifies a new CRABP1-binding ligand C32. Using the P19-MN differentiation system, the study establishes C32 and previously reported C4 as CRABP1 ligands that can modulate CaMKII activation in the P19-MN differentiation process. Further, in committed MN cells, elevating CRABP1 reduces excitotoxicity-triggered MN death, supporting a protective role for CRABP1 signaling in MN survival. C32 and C4 CRABP1 ligands were also protective against excitotoxicity-triggered MN death. The results provide insight into the potential of signaling pathway-selective, CRABP1-binding, atRA-like ligands in mitigating MN degenerative diseases.
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Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Neuronas Motoras , Degeneración Nerviosa , Receptores de Ácido Retinoico , Tretinoina , Animales , Ratones , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Receptores de Ácido Retinoico/metabolismo , Tretinoina/metabolismo , Neuronas Motoras/patologíaRESUMEN
Elaboration of tumor necrosis factor (TNF) is a very early event in development of ischemia/reperfusion injury pathophysiology. Therefore, TNF may be a prominent mediator of endothelial cell and vascular wall dysfunction in sickle cell anemia, a hypothesis we addressed using NY1DD, S+SAntilles , and SS-BERK sickle transgenic mice. Transfusion experiments revealed participation of abnormally activated blood monocytes exerting an endothelial activating effect, dependent upon Egr-1 in both vessel wall and blood cells, and upon NFκB(p50) in a blood cell only. Involvement of TNF was identified by beneficial impact from TNF blockers, etanercept and infliximab, with less benefit from an IL-1 blocker, anakinra. In therapeutic studies, etanercept ameliorated multiple disturbances of the murine sickle condition: monocyte activation, blood biomarkers of inflammation, low platelet count and Hb, vascular stasis triggered by hypoxia/reoxygenation (but not if triggered by hemin infusion), tissue production of neuro-inflammatory mediators, endothelial activation (monitored by tissue factor and VCAM-1 expression), histopathologic liver injury, and three surrogate markers of pulmonary hypertension (perivascular inflammatory aggregates, arteriolar muscularization, and right ventricular mean systolic pressure). In aggregate, these studies identify a prominent-and possibly dominant-role for an abnormal monocyte-TNF-endothelial activation axis in the sickle context. Its presence, plus the many benefits of etanercept observed here, argue that pilot testing of TNF blockade should be considered for human sickle cell anemia, a challenging but achievable translational research goal.
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Anemia de Células Falciformes/metabolismo , Células Endoteliales/metabolismo , Monocitos/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Anemia de Células Falciformes/diagnóstico , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/genética , Animales , Anticuerpos Monoclonales/farmacología , Biomarcadores , Trasplante de Médula Ósea , Agregación Celular/genética , Agregación Celular/inmunología , Modelos Animales de Enfermedad , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Endotelio Vascular/metabolismo , Etanercept/farmacología , Etanercept/uso terapéutico , Pruebas de Función Cardíaca , Humanos , Mediadores de Inflamación , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Terapia Molecular Dirigida , Monocitos/efectos de los fármacos , Monocitos/inmunología , FN-kappa B/deficiencia , FN-kappa B/genética , Fenotipo , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Tromboplastina/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited â¼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4ß1, or αVß3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4(-/-) mice transplanted with TLR4(+/+) sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4(-/-) ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.
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Anemia de Células Falciformes/metabolismo , Células Endoteliales/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Vasoconstricción , Animales , Células de la Médula Ósea/citología , Adhesión Celular , Haptoglobinas/metabolismo , Hemo/química , Hemoglobinas/química , Hemólisis , Hemopexina/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación , Lipopolisacáridos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Subunidad p50 de NF-kappa B/metabolismo , Estrés Oxidativo , Fenotipo , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: A motor unit (MU) is formed by a single alpha motor neuron (MN) and the muscle fibers it innervates. The MU is essential for all voluntary movements. Functional deficits in the MU result in neuromuscular disorders (NMDs). The pathological mechanisms underlying most NMDs remain poorly understood, in part due to the lack of in vitro models that can comprehensively recapitulate multistage intercellular interactions and physiological function of the MU. RESULTS: We have designed a novel three-dimensional (3D) bilayer hydrogel tri-culture system where architecturally organized MUs can form in vitro. A sequential co-culture procedure using the three cell types of a MU, MN, myoblast, and Schwann cell was designed to construct a co-differentiating tri-culture on a bilayer hydrogel matrix. We utilized a µ-molded hydrogel with an additional Matrigel layer to form the bilayer hydrogel device. The µ-molded hydrogel layer provides the topological cues for myoblast differentiation. The Matrigel layer, with embedded Schwann cells, not only separates the MNs from myoblasts but also provides a proper micro-environment for MU development. The completed model shows key MU features including an organized MU structure, myelinated nerves, aligned myotubes innervated on clustered neuromuscular junctions (NMJs), MN-driven myotube contractions, and increases in cytosolic Ca2+ upon stimulation. CONCLUSIONS: This organized and functional in vitro MU model provides an opportunity to study pathological events involved in NMDs and peripheral neuropathies, and can serve as a platform for physiological and pharmacological studies such as modeling and drug screening. Technically, the rational of this 3D bilayer hydrogel co-culture system exploits multiple distinct properties of hydrogels, facilitating effective and efficient co-culturing of diverse cell types for tissue engineering.
RESUMEN
Background Microparticles and endothelial microparticles (EMPs) are implicated in accelerating cardiovascular disease (CVD); however, data in pediatrics are limited. We examined the relationship of microparticles and EMPs with adiposity and subclinical CVD risk measures in a pediatric population to determine their potential as biomarkers of CVD risk. Methods and Results A cross-sectional study of youth (n=280; ages 8-20 years) with a range of body mass index categories was used. Microparticles, EMPs, and activated EMPs were measured by flow cytometry. %Body fat and %visceral adipose tissue were measured by dual X-ray absorptiometry. Measures of arterial stiffness and vascular wall structure were obtained. Linear regression (with log-transformed outcomes) and logistic regression were used to evaluate associations and all results were exponentiated. Youth with overweight/obesity and severe obesity had 2.50 (95% CI, 1.56-4.01) and 3.42 (95% CI, 2.15-5.43) times the geometric means of the total number of microparticles, respectively, compared with those with normal weight. Youth with overweight/obesity and severe obesity had 1.97 (95% CI, 1.09-3.55) and 2.34 (95% CI, 1.31-4.19) times the geometric means of the total number of EMPs, respectively, compared with those with normal weight. There were positive associations between the levels of both microparticles and EMPs with higher adiposity measures and poor CVD risk measures. Youth with higher adiposity showed 1.84 times the odds of having high levels of activated EMPs (%) (odds ratio, 1.84; 95% CI, 1.08-3.14) compared with those with normal weight. Conclusions Levels of microparticles, EMPs, and activated EMPs were positively associated with adiposity and poor subclinical CVD risk in a pediatric population.
Asunto(s)
Enfermedades Cardiovasculares , Micropartículas Derivadas de Células , Obesidad Mórbida , Humanos , Adolescente , Niño , Sobrepeso , Estudios Transversales , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/etiología , Endotelio Vascular , Obesidad/complicaciones , Obesidad/diagnóstico , Obesidad/epidemiologíaRESUMEN
Much of the bacterial anticancer therapy being developed relies on the ability of bacteria to specifically colonise tumours. Initial attempts to translate promising Salmonella enterica Typhimurium (S. Typhimurium) preclinical results to the clinical setting failed, primarily due to lack of tumour colonisation and the significant toxicities from systemically administered Gram-negative bacteria. To address the difference in results between preclinical experiments performed in mice with transplant tumours and clinical trials in human volunteers with autochthonous tumours, a genetically engineered mouse model of breast cancer (BALB-neuT) was utilised to develop a strain of virulence-attenuated S. Typhimurium capable of robust colonisation of autochthonous tumours. Several genes that code for bacterial surface molecules, responsible for signalling a toxic immune response against the bacteria, were mutated. The resulting S. Typhimurium strain, BCT2, allowed non-toxic intravenous administration of 3 × 106 colony forming units of bacteria in tumour-burdened mice when combined with a vascular disruption agent to induce intratumoral necrotic space and facilitate bacterial colonisation.
Asunto(s)
Ingeniería Genética , Neoplasias Mamarias Experimentales/microbiología , Salmonella typhimurium/fisiología , Animales , Femenino , Neoplasias Mamarias Experimentales/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidadRESUMEN
The ultimate goal of bacterial based cancer therapy is to achieve non-toxic penetration and colonisation of the tumour microenvironment. To overcome this efficacy-limiting toxicity of anticancer immunotherapy, we have tested a therapy comprised of systemic delivery of a vascular disrupting agent to induce intratumoral necrotic space, cannabidiol to temporarily inhibit angiogenesis and acute inflammation, and a strain of Salmonella Typhimurium that was engineered for non-toxic colonisation and expression of immunomodulators within the tumour microenvironment. This combination treatment strategy was administered to transgenic mice burdened with autochthonous mammary gland tumours and demonstrated a statistically significant 64% slower tumour growth and a 25% increase in mean survival time compared to control animals without treatment. These experiments were accomplished with minimal toxicity as measured by less than 7% weight loss and a return to normal weight gain within three days following intravenous administration of the bacteria. Thus, non-toxic, robust colonisation of the microenvironment was achieved to produce a significant antitumor effect.
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Bioingeniería/métodos , Neoplasias de la Mama/terapia , Modelos Animales de Enfermedad , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/biosíntesis , Salmonella typhimurium/metabolismo , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Factores Inmunológicos/síntesis química , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Salmonella typhimurium/química , Salmonella typhimurium/inmunología , Tasa de Supervivencia , Carga Tumoral/efectos de los fármacos , Carga Tumoral/fisiología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/fisiología , Virulencia/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Activation of the coagulation system is a characteristic feature of sickle cell anemia, which also includes clinical thrombosis. The sickle transgenic mouse abnormally expresses tissue factor (TF) on the pulmonary vein endothelium. Knowing that this aberrancy is stimulated by inflammation, we sought to determine whether nitric oxide (NO) contributes to regulation of endothelial TF expression in the sickle mouse model. We used the NY1DD sickle mouse, which exhibits a low-TF to high-TF phenotype switch on exposure to hypoxia/reoxygenation. Manipulations of NO biology, such as breathing NO or addition of arginine or L-NAME (N-nitro-L-arginine-methyl-ester) to the diet, caused significant modulations of TF expression. This was also seen in hBERK1 sickle mice, which have a different genetic background and already have high-TF even at ambient air. Study of NY1DD animals bred to overexpress endothelial nitric oxide synthase (eNOS; eNOS-Tg) or to have an eNOS knockout state (one eNOS(-/-) animal and several eNOS(+/-) animals) demonstrated that eNOS modulates endothelial TF expression in vivo by down-regulating it. Thus, the biodeficiency of NO characteristic of patients with sickle cell anemia may heighten risk for activation of the coagulation system.
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Anemia de Células Falciformes/metabolismo , Células Endoteliales/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Tromboplastina/metabolismo , Animales , Células Cultivadas , Ratones , Ratones Noqueados , Ratones TransgénicosRESUMEN
Altered inflammation signaling within the cerebral vasculature may be an important risk factor for stroke in children with sickle cell anemia (SCA). This study examines how differential expression of NFkappaB/p65 (RelA), KLF2, and other transcription factors may act as switches in inflammation signaling leading to observed differences between non-SCA (NS) African Americans and African Americans with SCA who are either at risk (AR) or not at risk (NAR) of childhood stroke based on occurrence of Circle of Willis disease. Clover/Transfac analysis was used to identify overrepresented transcription factor binding motifs on genes associated with inflammation. Transcription factor binding motifs for the NFkappaB family and RFX1 were overrepresented on inflammation signaling gene set analysis. Variations in protein expression were determined by flow cytometry of blood outgrowth endothelial cells (BOECs) from NS, AR, and NAR donors and Western blots of protein extracts from both unstimulated and TNFalpha/IL1beta-stimulated BOECs. BOECs from patients with SCA had more cytoplasmic-derived RelA compared with NS BOECs. Sickle BOECs also had heightened responses to inflammatory stimuli compared with NS BOECs, as shown by increased nuclear RelA, and intracellular adhesion molecule (ICAM) response to TNFalpha/IL1beta stimulation. Multiple control points in RelA signaling were associated with risk of childhood stroke. The ratio of proinflammatory factor RelA to anti-inflammatory factor KLF2 was greater in BOECs from AR donors than NS donors. Group risk of childhood stroke with SCA was greatest among individuals who exhibited increased expression of proinflammatory transcription factors and decreased expression of transcription factors that suppress inflammation.
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Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/metabolismo , Inflamación/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Accidente Cerebrovascular/etiología , Factor de Transcripción ReIA/metabolismo , Adolescente , Western Blotting , Estudios de Casos y Controles , Niño , Preescolar , Círculo Arterial Cerebral/patología , Células Endoteliales/clasificación , Células Endoteliales/metabolismo , Citometría de Flujo , Humanos , Inflamación/complicaciones , Factores de Riesgo , Adulto JovenRESUMEN
Aim: We assessed reproducibility of endothelial microparticles (EMPs) enumeration among youth. Methods & results: Four microparticle (MP) indices - total MP per microliter platelet free plasma (PFP), total EMPs per microliter PFP, percent activated EMPs and percent lactadherin positive (LACT[+]) of total EMPs - were measured at two visits (baseline and 7 ± 3 days follow-up) to determine reproducibility overall and by obesity status. We examined CD31+ or CD144+ with CD41-EMP events of size 0.3-1.0 µm. No statistically significant differences were observed between visits for any of the four MP indices. The within-participant and between-participant coefficient of variation was acceptable (range: 1.13-2.37) with good intraclass-correlation coefficient for all indices except total MP per microliter (range: 0.10-1.00). Conclusion: Total EMPs per microliter PFP, percent-activated EMPs and percent LACT(+) of total EMPs are reproducible among youth.
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Plaquetas/citología , Micropartículas Derivadas de Células/química , Endotelio Vascular/citología , Obesidad/diagnóstico , Adolescente , Biomarcadores/metabolismo , Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Niño , Estudios Transversales , Endotelio Vascular/metabolismo , Femenino , Humanos , Masculino , Obesidad/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Background We examined feasibility of a unique approach towards gaining insight into heritable risk for early atherosclerosis: surveying gene expression by endothelial cells from living subjects. Methods and Results Subjects aged <50 years (mean age, 37; range, 22-49) without obstructive coronary artery disease underwent coronary reactivity testing that identified them as having normal or abnormal coronary endothelial function. Cultures of Blood Outgrowth Endothelial Cells (BOEC) from 6 normal and 13 abnormal subjects passed rigorous quality control and were used for microarray assessment of gene expression. Of 9 genes differentially expressed at false discovery rate <0.1%, we here focus upon abnormal subjects having elevated expression of HMGB1 (high mobility group box 1) which we unexpectedly found to be linked to low LAMC1 (laminin gamma 1) expression. This linkage was corroborated by 3 of our past studies and confirmed bio-functionally. Compared with normal BOEC, abnormal BOEC released 13±3-fold more HMGB1 in response to lipopolysaccharide; and they deposited one tenth as much LAMC1 into collagen subendothelial matrix during culture. Clinical follow-up data are provided for 4 normal subjects (followed 13.4±0.1 year) and for 12 abnormal subjects (followed 9.1±4.5 years). Conclusions The known pathogenic effects of high-HMGB1 and low-LAMC1 predict that the combination would biologically converge upon the focal adhesion complex, to the detriment of endothelial shear responsiveness. This gene expression pattern may comprise a heritable risk state that promotes early coronary atherosclerosis. If so, the testing could be applied even in childhood, enabling early intervention. This approach offers a way to bridge the information gap between genetics and clinical phenotype.
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Enfermedad de la Arteria Coronaria/metabolismo , Células Endoteliales/metabolismo , Proteína HMGB1/metabolismo , Laminina/metabolismo , Adulto , Enfermedad de la Arteria Coronaria/genética , Estudios de Factibilidad , Femenino , Estudios de Seguimiento , Expresión Génica , Perfilación de la Expresión Génica , Proteína HMGB1/genética , Humanos , Laminina/genética , Masculino , Persona de Mediana Edad , Cultivo Primario de Células , Medición de Riesgo , Adulto JovenRESUMEN
We have investigated a unique cell type, blood outgrowth endothelial cells (BOEC), as a cell-based gene therapy approach to pulmonary hypertension. BOEC are bona fide endothelial cells, obtained from peripheral blood, that can be expanded to vast numbers, and are amenable to both cryopreservation and genetic modification. We established primary cultures of rat BOEC and genetically altered them to over-express human eNOS plus green fluorescent protein (rBOEC/eNOS) or to express GFP only (rBOEC/GFP). We gave monocrotaline to rats on day 0, and they developed severe pulmonary hypertension. As a Prevention model, we infused saline or rBOEC/GFP or rBOEC/eNOS on day 3, and then examined endpoints on day 24. The rBOEC/eNOS recipients developed elevated NOx (serum and lung) and less severe: elevation of right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and pulmonary arteriolar muscularization and loss of alveolar density. As an Intervention model, we waited until day 21 to give the test infusions, and we examined endpoints on day 35. The rBOEC/eNOS recipients again developed elevated NOx and manifested the same improvements. Indeed, rBOEC/eNOS infusion not only prevented worsening of RVSP but also partially reversed established arteriolar muscularization. These data suggest that BOEC may be useful as a carrier cell for genetic strategies targeting pulmonary hypertension. Their properties render BOEC amenable to preclinical and scale-up studies, available for autologous therapies, and tolerant of modification and storage for potential future use in patients at risk for PAH, eg, as defined by genetics or medical condition.
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Células Endoteliales/enzimología , Terapia Genética , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/terapia , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Modelos Animales de Enfermedad , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Hipertensión Pulmonar/prevención & control , Ratas Endogámicas F344 , Trasplante AutólogoRESUMEN
Since the available microarray data of BOEC (human blood outgrowth endothelial cells), large vessel, and microvascular endothelial cells were from two different platforms, a working cross-platform normalization method was needed to make these data comparable. With six HUVEC (human umbilical vein endothelial cells) samples hybridized on two-channel cDNA arrays and six HUVEC samples on Affymetrix arrays, 64 possible combinations of a three-step normalization procedure were investigated to search for the best normalization method, which was selected, based on two criteria measuring the extent to which expression profiles of biological samples of the same cell type arrayed on two platforms were indistinguishable. Next, three discriminative gene lists between the large vessel and the microvascular endothelial cells were achieved by SAM (significant analysis of microarrays), PAM (prediction analysis for microarrays), and a combination of SAM and PAM lists. The final discriminative gene list was selected by SVM (support vector machine). Based on this discriminative gene list, SVM classification analysis with best tuning parameters and 10,000 times of validations showed that BOEC were far from large vessel cells, they either formed their own class, or fell into the microvascular class. Based on all the common genes between the two platforms, SVM analysis further confirmed this conclusion.
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Vasos Sanguíneos/citología , Células Endoteliales/citología , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Vasos Sanguíneos/fisiología , Células Cultivadas , Análisis por Conglomerados , Sondas de ADN/análisis , Bases de Datos de Ácidos Nucleicos , Análisis Discriminante , Células Endoteliales/fisiología , Humanos , ARN/análisis , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Venas Umbilicales/citología , Venas Umbilicales/fisiologíaRESUMEN
Sickle cell anemia is accompanied by the activation of coagulation and thrombosis. We have studied the abnormal expression of tissue factor (TF) by the pulmonary vein endothelium of the mild-phenotype NY1DD sickle transgenic. As detected by immunofluorescence microscopy, this occurs only after the NY1DD mouse is exposed to hypoxia/reoxygenation (H/R), which actually causes ischemia/reperfusion in the sickle cell disease-but not the normal-mouse model. We tested the hypothesis that the nuclear factor-kappa B (NFkappaB)-activating inflammation that develops in post-H/R NY1DD mice is responsible for this phenotype switch. Various NFkappaB inhibitors (including p50-specific andrographolide) demonstrated that endothelial TF positivity is NFkappaB dependent. Several systemic inflammatory stimulators (tumor necrosis factor [TNFalpha], lipopolysaccharide, thioglycollate, and carageenan) given to control mice showed that the inflammatory promotion of TF expression by only pulmonary vein endothelium is not specific to the sickle cell disease model. We bred the NFkappaB(p50)-/- state into the NY1DD mouse. Combined with marrow transplantation, this allowed the creation of NY1DD mice that were NFkappaB(p50)-/- only in peripheral blood cells (and marrow) versus only in vessel walls (and tissues). This process revealed that endothelial TF expression in the NY1DD mouse is highly dependent on NFkappaB(p50) in peripheral blood mononuclear cells-but not in the vessel wall. In confirmation, the infusion of post-H/R sickle mouse blood mononuclear cells into naïve NY1DD mice stimulated endothelial TF expression; the infusion of such cells from unstimulated sickle cell disease mice at ambient air did not stimulate TF expression. We conclude that peripheral blood mononuclear cells indirectly promote endothelial TF expression via a NFkappaB(p50)-dependent mechanism. This approach may be relevant to the role of coagulopathy in clinical sickle cell disease.
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Anemia de Células Falciformes/sangre , Coagulación Sanguínea/fisiología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Leucocitos Mononucleares/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Tromboplastina/metabolismo , Anemia de Células Falciformes/complicaciones , Animales , Técnicas de Inactivación de Genes , Humanos , Inflamación/metabolismo , Inflamación/patología , Leucocitos Mononucleares/patología , Ratones , Ratones Transgénicos , Subunidad p50 de NF-kappa B/antagonistas & inhibidores , Subunidad p50 de NF-kappa B/deficienciaRESUMEN
Human blood outgrowth endothelial cells (hBOECs) may be useful delivery cells for gene therapy. hBOECs have high expansion capacity and a stable phenotype. If incorporated into blood vessels, hBOECs could release therapeutic agents directly into the bloodstream. However, little is known about the lodging and homing of hBOECs in vivo. We examined the homing patterns of hBOECs in mice and explored extending cell-based factor VIII (FVIII) gene therapy from mice to larger animals. hBOECs were injected into NOD/SCID mice to determine where they localize, how localization changes over time, and if there were toxic effects on host organs. The presence of hBOECs in mouse organs was determined by quantitative polymerase chain reaction (qPCR) and immunofluorescence microscopy. hBOECs lodged most notably in mouse lungs at 3 h, but by 24 h, no differences were observed among 9 organs. The longevity of hBOECs was assessed up to 7 months in vivo. hBOECs expanded well and then reached a plateau in vivo. hBOECs from older cultures expanded equally well in vivo as younger hBOECs. hBOECs caused no noticeable organ toxicity up to 3 days after injection. When mice were pretreated with antibodies to E-selectin, P-selectin, or anti-alpha4 integrin prior to hBOEC injection, the number of hBOECs in lungs at 3 h was decreased. Preliminary studies that infused hemophilic dogs with autologous canine BOECs that overexpressed FVIII (B-domain deleted) showed improvement in whole blood clotting times (WBCTs). In conclusion, the survivability, expandability, and lack of toxicity of BOECs in vivo indicate that they may be valuable host cells for gene therapy.
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Endotelio Vascular/citología , Factor VIII/genética , Terapia Genética , Animales , Secuencia de Bases , Cartilla de ADN , Perros , Endotelio Vascular/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microscopía Fluorescente , Reacción en Cadena de la PolimerasaRESUMEN
Genetic differences in endothelial biology could underlie development of phenotypic heterogeneity among persons afflicted with vascular diseases. We obtained blood outgrowth endothelial cells from 20 subjects with sickle cell anemia (age, 4-19 years) shown to be either at-risk (n=11) or not-at-risk (n=9) for ischemic stroke because of, respectively, having or not having occlusive disease at the circle of Willis. Gene expression profiling identified no significant single gene differences between the 2 groups, as expected. However, analysis of Biological Systems Scores, using gene sets that were predetermined to survey each of 9 biologic systems, showed that only changes in inflammation signaling are characteristic of the at-risk subjects, as supported by multiple statistical approaches. Correspondingly, subsequent biologic testing showed significantly exaggerated RelA activation on the part of blood outgrowth endothelial cells from the at-risk subjects in response to stimulation with interleukin-1beta/tumor necrosis factoralpha. We conclude that the pathobiology of circle of Willis disease in the child with sickle cell anemia predominantly involves inflammation biology, which could reflect differences in genetically determined endothelial biology that account for differing host responses to inflammation.
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Anemia de Células Falciformes/genética , Círculo Arterial Cerebral , Endotelio Vascular , Regulación de la Expresión Génica , Accidente Cerebrovascular/genética , Factor de Transcripción ReIA/genética , Adolescente , Adulto , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/metabolismo , Anemia de Células Falciformes/patología , Células Cultivadas , Niño , Preescolar , Círculo Arterial Cerebral/metabolismo , Círculo Arterial Cerebral/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/farmacología , Masculino , Factores de Riesgo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Endothelial cells are an attractive vehicle for gene therapy because they may be used in an autologous fashion and may allow for direct exposure of the gene product into the intravascular space. To explore this future potential, a reproducible system was developed for the culture of murine blood outgrowth endothelial cells. These cells demonstrated acetylated low-density lipoprotein (LDL) incorporation, matrigel tube formation, and specific endothelial staining characteristics, namely P1H12, VeCAD, vascular cell adhesion molecule (VCAM), vWF, platelet endothelial cell adhesion molecule (PECAM-1), and vascular endothelial growth factor receptor-2 (VEGFR2). They were also negative for smooth muscle actin and monocytic markers CD11b, CD14, and CD16. Moreover, these cells were amendable to gene transfer with red fluorescent and green fluorescent expression vectors as well as human Factor VIII (hFVIII) while maintaining endothelial characteristics. Both source- and gene-introduced cells also manifested excellent proliferative potential. Furthermore, murine blood outgrowth endothelial cells (BOECs) demonstrated persistent in vivo seeding in the liver, lung, spleen, and bone morrow of recipient mice.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Endotelio Vascular/citología , Factor VIII/genética , Terapia Genética/métodos , Hemofilia A/terapia , Animales , Biomarcadores , División Celular , Trasplante de Células/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Mutantes , Ratones SCID , Plásmidos , Talasemia/terapia , Proteína Fluorescente RojaRESUMEN
Abnormal tissue factor (TF) expression has been demonstrated on blood monocytes and circulating endothelial cells in humans with sickle cell anemia. We have now studied sickle transgenic mice to help define the biology of endothelial TF expression in sickle disease. Using immunostaining of tissue sections, we find that this is confined almost exclusively to the pulmonary veins. About 15% and 13% of these exhibit TF-positive endothelium in the wild-type normal mouse and the normal human hemoglobin (HbA)-expressing control transgenic mouse, respectively. The mild sickle mouse is indistinguishable from normal (approximately 14% positive), but TF expression is significantly elevated in the moderate and severe mouse models of sickle disease (approximately 29% and approximately 41% positive, respectively). Exposure of the mild sickle mouse to hypoxia for 3 hours, followed by reoxygenation, converted its TF expression phenotype to that of the severe sickle mouse (approximately 36% positive). Pretreatment with lovastatin eliminated excessive expression of TF in the posthypoxic mild sickle mouse (approximately 16% positive) and in the more severe mouse at ambient air (approximately 21% positive). In addition to identifying tissue expression of endothelial TF in the sickle lung, these studies implicate reperfusion injury physiology in its expression and suggest a rationale for use of statins in sickle disease.