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1.
Cell ; 184(17): 4547-4563.e17, 2021 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-34314701

RESUMEN

Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.


Asunto(s)
Cerebro/patología , Proteína 4 Similar a ELAV/genética , Ácido Glutámico/metabolismo , Mutación/genética , Neuronas/patología , Organoides/metabolismo , Empalme del ARN/genética , Proteínas tau/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Biomarcadores/metabolismo , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Muerte Celular/efectos de los fármacos , Línea Celular , Humanos , Hidrazonas/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Morfolinas/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Organoides/efectos de los fármacos , Organoides/ultraestructura , Fosforilación/efectos de los fármacos , Pirimidinas/farmacología , Empalme del ARN/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Gránulos de Estrés/efectos de los fármacos , Gránulos de Estrés/metabolismo , Sinapsis/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética
2.
Kidney Int ; 102(2): 293-306, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35469894

RESUMEN

Recent epidemiological studies suggest that some patients with diabetes progress to kidney failure without significant albuminuria and glomerular injury, suggesting a critical role of kidney tubular epithelial cell (TEC) injury in diabetic kidney disease (DKD) progression. However, the major risk factors contributing to TEC injury and progression in DKD remain unclear. We previously showed that expression of endoplasmic reticulum-resident protein Reticulon-1A (RTN1A) increased in human DKD, and the increased RTN1A expression promoted TEC injury through endoplasmic reticulum (ER) stress response. Here, we show that TEC-specific RTN1A overexpression worsened DKD in mice, evidenced by enhanced tubular injury, tubulointerstitial fibrosis, and kidney function decline. But RTN1A overexpression did not exacerbate diabetes-induced glomerular injury or albuminuria. Notably, RTN1A overexpression worsened both ER stress and mitochondrial dysfunction in TECs under diabetic conditions by regulation of ER-mitochondria contacts. Mechanistically, ER-bound RTN1A interacted with mitochondrial hexokinase-1 and the voltage-dependent anion channel-1 (VDAC1), interfering with their association. This disengagement of VDAC1 from hexokinase-1 resulted in activation of apoptotic and inflammasome pathways, leading to TEC injury and loss. Thus, our observations highlight the importance of ER-mitochondrial crosstalk in TEC injury and the salient role of RTN1A-mediated ER-mitochondrial contact regulation in DKD progression.


Asunto(s)
Diabetes Mellitus , Nefropatías Diabéticas , Retículo Endoplásmico , Mitocondrias , Proteínas del Tejido Nervioso , Albuminuria/metabolismo , Animales , Apoptosis , Diabetes Mellitus/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Células Epiteliales/metabolismo , Hexoquinasa/metabolismo , Humanos , Ratones , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/genética
3.
Mod Pathol ; 34(8): 1456-1467, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33795830

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated clinical syndrome COVID-19 are causing overwhelming morbidity and mortality around the globe and disproportionately affected New York City between March and May 2020. Here, we report on the first 100 COVID-19-positive autopsies performed at the Mount Sinai Hospital in New York City. Autopsies revealed large pulmonary emboli in six cases. Diffuse alveolar damage was present in over 90% of cases. We also report microthrombi in multiple organ systems including the brain, as well as hemophagocytosis. We additionally provide electron microscopic evidence of the presence of the virus in our samples. Laboratory results of our COVID-19 cohort disclose elevated inflammatory markers, abnormal coagulation values, and elevated cytokines IL-6, IL-8, and TNFα. Our autopsy series of COVID-19-positive patients reveals that this disease, often conceptualized as a primarily respiratory viral illness, has widespread effects in the body including hypercoagulability, a hyperinflammatory state, and endothelial dysfunction. Targeting of these multisystemic pathways could lead to new treatment avenues as well as combination therapies against SARS-CoV-2 infection.


Asunto(s)
COVID-19/fisiopatología , Pulmón/fisiopatología , Embolia Pulmonar/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Autopsia , Coagulación Sanguínea , COVID-19/sangre , COVID-19/patología , COVID-19/virología , Causas de Muerte , Citocinas/sangre , Femenino , Interacciones Huésped-Patógeno , Humanos , Mediadores de Inflamación/sangre , Pulmón/patología , Pulmón/virología , Masculino , Persona de Mediana Edad , Ciudad de Nueva York , Embolia Pulmonar/sangre , Embolia Pulmonar/patología , Embolia Pulmonar/virología , SARS-CoV-2/patogenicidad
4.
J Am Soc Nephrol ; 31(10): 2372-2391, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32737144

RESUMEN

BACKGROUND: Maintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood. METHODS: Cells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside-induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine Nebl expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes. RESULTS: Nebl knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation. CONCLUSIONS: LIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments.


Asunto(s)
Actinas/fisiología , Filamentos Intermedios/fisiología , Enfermedades Renales/patología , Glomérulos Renales/patología , Podocitos/patología , Vimentina/fisiología , Animales , Técnicas de Cultivo de Célula , Proteínas del Citoesqueleto/fisiología , Humanos , Enfermedades Renales/etiología , Proteínas con Dominio LIM/fisiología , Ratones , Ratas
5.
J Cell Mol Med ; 24(13): 7214-7227, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32485073

RESUMEN

Extracellular vesicles (EVs) have recently emerged as an important carrier for various genetic materials including microRNAs (miRs). Growing evidences suggested that several miRs transported by EVs were particularly involved in modulating cardiac function. However, it has remained unclear what miRs are enriched in EVs and play an important role in the pathological condition. Therefore, we established the miR expression profiles in EVs from murine normal and failing hearts and consecutively identified substantially altered miRs. In addition, we have performed bioinformatics approach to predict potential cardiac outcomes through the identification of miR targets. Conclusively, we observed approximately 63% of predicted targets were validated with previous reports. Notably, the predicted targets by this approach were often involved in both beneficial and malicious signalling pathways, which may reflect heterogeneous cellular origins of EVs in tissues. Lastly, there has been an active debate on U6 whether it is a proper control. Through further analysis of EV miR profiles, miR-676 was identified as a superior reference control due to its consistent and abundant expressions. In summary, our results contribute to identifying specific EV miRs for the potential therapeutic targets in heart failure and suggest that miR-676 as a new reference control for the EV miR studies.


Asunto(s)
Vesículas Extracelulares/genética , Perfilación de la Expresión Génica , Insuficiencia Cardíaca/genética , MicroARNs/genética , Animales , Regulación hacia Abajo/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestructura , Masculino , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Proteómica , Reproducibilidad de los Resultados , Regulación hacia Arriba/genética
6.
J Med Virol ; 92(7): 699-702, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32314810

RESUMEN

Neurologic sequelae can be devastating complications of respiratory viral infections. We report the presence of virus in neural and capillary endothelial cells in frontal lobe tissue obtained at postmortem examination from a patient infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Our observations of virus in neural tissue, in conjunction with clinical correlates of worsening neurologic symptoms, pave the way to a closer understanding of the pathogenic mechanisms underlying central nervous system involvement by SARS-CoV-2.


Asunto(s)
Ageusia/diagnóstico , Ataxia/diagnóstico , Betacoronavirus/patogenicidad , Infecciones por Coronavirus/diagnóstico , Trastornos del Olfato/diagnóstico , Neumonía Viral/diagnóstico , Convulsiones/diagnóstico , Anciano , Ageusia/complicaciones , Ageusia/fisiopatología , Ageusia/virología , Ataxia/complicaciones , Ataxia/fisiopatología , Ataxia/virología , Betacoronavirus/genética , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/complicaciones , Infecciones por Coronavirus/fisiopatología , Infecciones por Coronavirus/virología , Células Endoteliales/patología , Células Endoteliales/virología , Resultado Fatal , Lóbulo Frontal/irrigación sanguínea , Lóbulo Frontal/patología , Lóbulo Frontal/virología , Hospitalización , Humanos , Pulmón/irrigación sanguínea , Pulmón/patología , Pulmón/virología , Masculino , Neuronas/patología , Neuronas/virología , Trastornos del Olfato/complicaciones , Trastornos del Olfato/fisiopatología , Trastornos del Olfato/virología , Pandemias , Neumonía Viral/complicaciones , Neumonía Viral/fisiopatología , Neumonía Viral/virología , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Convulsiones/complicaciones , Convulsiones/fisiopatología , Convulsiones/virología
7.
Eur Heart J ; 40(12): 967-978, 2019 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-29668883

RESUMEN

AIMS: Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. There is currently no information on the role of the delta-like homologue 1 (Dlk1) in the regulation of the fibrotic response. Here, we investigated whether Dlk1 is involved in cardiac fibroblast-to-myofibroblast differentiation and regulates myocardial fibrosis and explored the molecular mechanism underpinning its effects in this process. METHODS AND RESULTS: Using Dlk1-knockout mice and adenoviral gene delivery, we demonstrate that overexpression of Dlk1 in cardio-fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process is mediated by TGF-ß1 signalling, since isolated fibroblasts lacking Dlk1 exhibited a higher activation of the TGF-ß1/Smad-3 pathway at baseline, leading to an earlier acquisition of a myofibroblast phenotype. Likewise, Dlk1-null mice displayed increased TGF-ß1/Smad3 cardiac activity, resulting in infiltration/accumulation of myofibroblasts, induction and deposition of extra-domain A-fibronectin isoform and collagen, and activation of pro-fibrotic markers. Furthermore, these profibrotic events were associated with disrupted myofibril integrity, myocyte hypertrophy, and cardiac dysfunction. Interestingly, Dlk1 expression was down-regulated in ischaemic human and porcine heart tissues. Mechanistically, miR-370 mediated Dlk1's regulation of cardiac fibroblast-myofibroblast differentiation by directly targeting TGFß-R2/Smad-3 signalling, while the Dlk1 canonical target, Notch pathway, does not seem to play a role in this process. CONCLUSION: These findings are the first to demonstrate an inhibitory role of Dlk1 of cardiac fibroblast-to-myofibroblast differentiation by interfering with TGFß/Smad-3 signalling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFß signalling leads to chronic fibrosis.


Asunto(s)
Proteínas de Unión al Calcio/genética , Fibroblastos/metabolismo , Fibrosis/genética , Miocardio/patología , Miofibroblastos/metabolismo , Animales , Diferenciación Celular , Regulación hacia Abajo , Humanos , Masculino , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Proteína smad3/genética , Porcinos , Factor de Crecimiento Transformador beta1/genética
8.
Development ; 143(12): 2160-71, 2016 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-27122169

RESUMEN

Primary cilia have been linked to signaling pathways involved in cell proliferation, cell motility and cell polarity. Defects in ciliary function result in developmental abnormalities and multiple ciliopathies. Patients affected by severe ciliopathies, such as Meckel syndrome, present several ocular surface disease conditions of unclear pathogenesis. Here, we show that primary cilia are predominantly present on basal cells of the mouse corneal epithelium (CE) throughout development and in the adult. Conditional ablation of cilia in the CE leads to an increase in proliferation and vertical migration of basal corneal epithelial cells (CECs). A consequent increase in cell density of suprabasal layers results in a thicker than normal CE. Surprisingly, in cilia-deficient CE, cilia-mediated signaling pathways, including Hh and Wnt pathways, were not affected but the intensity of Notch signaling was severely diminished. Although Notch1 and Notch2 receptors were expressed normally, nuclear Notch1 intracellular domain (N1ICD) expression was severely reduced. Postnatal development analysis revealed that in cilia-deficient CECs downregulation of the Notch pathway precedes cell proliferation defects. Thus, we have uncovered a function of the primary cilium in maintaining homeostasis of the CE by balancing proliferation and vertical migration of basal CECs through modulation of Notch signaling.


Asunto(s)
Cilios/metabolismo , Epitelio Corneal/metabolismo , Homeostasis , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Transducción de Señal , Animales , Animales Recién Nacidos , Movimiento Celular , Proliferación Celular , Cilios/ultraestructura , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Epitelio Corneal/ultraestructura , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , Proteínas Supresoras de Tumor/metabolismo , Cicatrización de Heridas
9.
J Biol Chem ; 292(51): 21137-21148, 2017 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-28982981

RESUMEN

Kidney podocytes represent a key constituent of the glomerular filtration barrier. Identifying the molecular mechanisms of podocyte injury and survival is important for better understanding and management of kidney diseases. KIBRA (kidney brain protein), an upstream regulator of the Hippo signaling pathway encoded by the Wwc1 gene, shares the pro-injury properties of its putative binding partner dendrin and antagonizes the pro-survival signaling of the downstream Hippo pathway effector YAP (Yes-associated protein) in Drosophila and MCF10A cells. We recently identified YAP as an essential component of the glomerular filtration barrier that promotes podocyte survival by inhibiting dendrin pro-apoptotic function. Despite these recent advances, the signaling pathways that mediate podocyte injury remain poorly understood. Here we tested the hypothesis that, similar to its role in other model systems, KIBRA promotes podocyte injury. We found increased expression of KIBRA and phosphorylated YAP protein in glomeruli of patients with biopsy-proven focal segmental glomerulosclerosis (FSGS). KIBRA/WWc1 overexpression in murine podocytes promoted LATS kinase phosphorylation, leading to subsequent YAP Ser-127 phosphorylation, YAP cytoplasmic sequestration, and reduction in YAP target gene expression. Functionally, KIBRA overexpression induced significant morphological changes in podocytes, including disruption of the actin cytoskeletal architecture and reduction of focal adhesion size and number, all of which were rescued by subsequent YAP overexpression. Conversely, constitutive KIBRA knockout mice displayed reduced phosphorylated YAP and increased YAP expression at baseline. These mice were protected from acute podocyte foot process effacement following protamine sulfate perfusion. KIBRA knockdown podocytes were also protected against protamine-induced injury. These findings suggest an important role for KIBRA in the pathogenesis of podocyte injury and the progression of proteinuric kidney disease.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Podocitos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Biopsia , Femenino , Regulación de la Expresión Génica , Glomeruloesclerosis Focal y Segmentaria/enzimología , Glomeruloesclerosis Focal y Segmentaria/patología , Células HEK293 , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosforilación , Podocitos/patología , Podocitos/ultraestructura , Procesamiento Proteico-Postraduccional , Interferencia de ARN , Serina/metabolismo , Factores de Transcripción , Proteínas Señalizadoras YAP
10.
J Virol ; 91(2)2017 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-27847357

RESUMEN

Virological synapses (VS) are adhesive structures that form between infected and uninfected cells to enhance the spread of HIV-1. During T cell VS formation, viral proteins are actively recruited to the site of cell-cell contact where the viral material is efficiently translocated to target cells into heterogeneous, protease-resistant, antibody-inaccessible compartments. Using correlative light and electron microscopy (CLEM), we define the membrane topography of the virus-containing compartments (VCC) where HIV is found following VS-mediated transfer. Focused ion beam scanning electron microscopy (FIB-SEM) and serial sectioning transmission electron microscopy (SS-TEM) were used to better resolve the fluorescent Gag-containing structures within the VCC. We found that small punctate fluorescent signals correlated with single viral particles in enclosed vesicular compartments or surface-localized virus particles and that large fluorescent signals correlated with membranous Gag-containing structures with unknown pathological function. CLEM imaging revealed distinct pools of newly deposited viral proteins within endocytic and nonendocytic compartments in VS target T cells. IMPORTANCE: This study directly correlates individual virus-associated objects observed in light microscopy with ultrastructural features seen by electron microscopy in the HIV-1 virological synapse. This approach elucidates which infection-associated ultrastructural features represent bona fide HIV protein complexes. We define the morphology of some HIV cell-to-cell transfer intermediates as true endocytic compartments and resolve unique synapse-associated viral structures created by transfer across virological synapses.


Asunto(s)
Linfocitos T CD4-Positivos/ultraestructura , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/virología , VIH-1/fisiología , VIH-1/ultraestructura , Interacciones Huésped-Patógeno , Humanos , Virión/fisiología , Internalización del Virus , Replicación Viral , Desencapsidación Viral
11.
Proc Natl Acad Sci U S A ; 111(3): 1078-83, 2014 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-24395808

RESUMEN

Therapeutic and diagnostic nanomaterials are being intensely studied for several diseases, including cancer and atherosclerosis. However, the exact mechanism by which nanomedicines accumulate at targeted sites remains a topic of investigation, especially in the context of atherosclerotic disease. Models to accurately predict transvascular permeation of nanomedicines are needed to aid in design optimization. Here we show that an endothelialized microchip with controllable permeability can be used to probe nanoparticle translocation across an endothelial cell layer. To validate our in vitro model, we studied nanoparticle translocation in an in vivo rabbit model of atherosclerosis using a variety of preclinical and clinical imaging methods. Our results reveal that the translocation of lipid-polymer hybrid nanoparticles across the atherosclerotic endothelium is dependent on microvascular permeability. These results were mimicked with our microfluidic chip, demonstrating the potential utility of the model system.


Asunto(s)
Aterosclerosis/fisiopatología , Sistemas de Liberación de Medicamentos , Endotelio/metabolismo , Nanopartículas del Metal/química , Animales , Aterosclerosis/tratamiento farmacológico , Modelos Animales de Enfermedad , Oro/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Imagen por Resonancia Magnética , Masculino , Microcirculación , Microfluídica , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Modelos Teóricos , Permeabilidad , Placa Aterosclerótica , Conejos , Resistencia al Corte
12.
Mol Med ; 21: 487-95, 2015 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-26062020

RESUMEN

Acute intermittent porphyria (AIP) is an autosomal-dominant hepatic disorder caused by the half-normal activity of hydroxymethylbilane (HMB) synthase. Symptomatic individuals experience life-threatening acute neurovisceral attacks that are precipitated by factors that induce the hepatic expression of 5-aminolevulinic acid synthase 1 (ALAS1), resulting in the marked accumulation of the putative neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Here, we provide the first detailed description of the biochemical and pathologic alterations in the explanted liver of an AIP patient who underwent orthotopic liver transplantation (OLT) due to untreatable and debilitating chronic attacks. After OLT, the recipient's plasma and urinary ALA and PBG rapidly normalized, and her attacks immediately stopped. In the explanted liver, (a) ALAS1 mRNA and activity were elevated approximately ~3- and 5-fold, and ALA and PBG concentrations were increased ~3- and 1,760-fold, respectively; (b) uroporphyrin III concentration was elevated; (c) microsomal heme content was sufficient, and representative cytochrome P450 activities were essentially normal; (d) HMB synthase activity was approximately half-normal (~42%); (e) iron concentration was slightly elevated; and (f) heme oxygenase I mRNA was increased approximately three-fold. Notable pathologic findings included nodular regenerative hyperplasia, previously not reported in AIP livers, and minimal iron deposition, despite the large number of hemin infusions received before OLT. These findings suggest that the neurovisceral symptoms of AIP are not associated with generalized hepatic heme deficiency and support the neurotoxicity of ALA and/or PBG. Additionally, they indicate that substrate inhibition of hepatic HMB synthase activity by PBG is not a pathogenic mechanism in acute attacks.


Asunto(s)
5-Aminolevulinato Sintetasa/genética , Hidroximetilbilano Sintasa/biosíntesis , Hígado/metabolismo , Porfiria Intermitente Aguda/genética , 5-Aminolevulinato Sintetasa/biosíntesis , Adulto , Ácido Aminolevulínico/sangre , Ácido Aminolevulínico/orina , Femenino , Hemo/metabolismo , Humanos , Hidroximetilbilano Sintasa/antagonistas & inhibidores , Hígado/patología , Trasplante de Hígado , Porfobilinógeno/sangre , Porfobilinógeno/orina , Porfiria Intermitente Aguda/enzimología , Porfiria Intermitente Aguda/patología , ARN Mensajero/biosíntesis , Uroporfirinas/metabolismo
13.
FASEB J ; 28(2): 644-54, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24174427

RESUMEN

Cardiac experimental biology and translational research would benefit from an in vitro surrogate for human heart muscle. This study investigated structural and functional properties and interventional responses of human engineered cardiac tissues (hECTs) compared to human myocardium. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs, >90% troponin-positive) were mixed with collagen and cultured on force-sensing elastomer devices. hECTs resembled trabecular muscle and beat spontaneously (1.18 ± 0.48 Hz). Microstructural features and mRNA expression of cardiac-specific genes (α-MHC, SERCA2a, and ACTC1) were comparable to human myocardium. Optical mapping revealed cardiac refractoriness with loss of 1:1 capture above 3 Hz, and cycle length dependence of the action potential duration, recapitulating key features of cardiac electrophysiology. hECTs reconstituted the Frank-Starling mechanism, generating an average maximum twitch stress of 660 µN/mm(2) at Lmax, approaching values in newborn human myocardium. Dose-response curves followed exponential pharmacodynamics models for calcium chloride (EC50 1.8 mM) and verapamil (IC50 0.61 µM); isoproterenol elicited a positive chronotropic but negligible inotropic response, suggesting sarcoplasmic reticulum immaturity. hECTs were amenable to gene transfer, demonstrated by successful transduction with Ad.GFP. Such 3-D hECTs recapitulate an early developmental stage of human myocardium and promise to offer an alternative preclinical model for cardiology research.


Asunto(s)
Miocardio/citología , Ingeniería de Tejidos/métodos , Línea Celular , Electrofisiología , Humanos
14.
Am J Dermatopathol ; 37(7): 563-6, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26091514

RESUMEN

The authors report 2 cases of measles demonstrating novel skin pathology that may be useful in establishing early diagnosis. Syncytial epithelial giant cells, which are characteristic of measles, were found to be present in the dermis, indicating that these cells are not specific to the lymphoid tissue and epithelia of which they are classically attributed to. The cells were not prominent, and required step sectioning to observe. These results were confirmed by electron microscopy, which showed virus capsid particles within the endoplasmic reticulum, secretory vesicles, and cytoplasm of multinucleated cells. One of the cases also demonstrated an unusual mixed infiltrate of eosinophils and fibrin thrombi, which has not been previously described. Both patients in this report recovered with supportive therapy.


Asunto(s)
Dermis/patología , Células Gigantes/ultraestructura , Sarampión/patología , Enfermedades Cutáneas Virales/patología , Cápside/ultraestructura , Dermis/ultraestructura , Femenino , Humanos , Masculino , Virus del Sarampión , Persona de Mediana Edad , Adulto Joven
15.
Development ; 137(5): 755-65, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20110314

RESUMEN

Dysfunction of the ureter often leads to urine flow impairment from the kidney to the bladder, causing dilation of the ureter and/or renal pelvis. Six1 is a crucial regulator of renal development: mutations in human SIX1 cause branchio-oto-renal (BOR) syndrome and Six1(-/-) mice exhibit renal agenesis, although the ureter is present. It remains unclear whether Six1 plays a role in regulating ureter morphogenesis. We demonstrate here that Six1 is differentially expressed during ureter morphogenesis. It was expressed in undifferentiated smooth muscle (SM) progenitors, but was downregulated in differentiating SM cells (SMCs) and had disappeared by E18.5. In Six1(-/-) mice, the ureteral mesenchymal precursors failed to condense and differentiate into normal SMCs and showed increased cell death, indicating that Six1 is required for the maintenance and normal differentiation of SM progenitors. A delay in SMC differentiation was observed in Six1(-/-) ureters. A lack of Six1 in the ureter led to hydroureter and hydronephrosis without anatomical obstruction when kidney formation was rescued in Six1(-/-) embryos by specifically expressing Six1 in the metanephric mesenchyme, but not the ureter, under control of the Eya1 promoter. We show that Six1 and Tbx18 genetically interact to synergistically regulate SMC development and ureter function and that their gene products form a complex in cultured cells and in the developing ureter. Two missense mutations in SIX1 from BOR patients reduced or abolished SIX1-TBX18 complex formation. These findings uncover an essential role for Six1 in establishing a functionally normal ureter and provide new insights into the molecular basis of urinary tract malformations in BOR patients.


Asunto(s)
Proteínas de Homeodominio/fisiología , Músculo Liso/embriología , Proteínas de Dominio T Box/fisiología , Uréter/embriología , Animales , Apoptosis/genética , Síndrome Branquio Oto Renal/embriología , Síndrome Branquio Oto Renal/genética , Diferenciación Celular/genética , Células Cultivadas , Embrión de Mamíferos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Hidronefrosis/embriología , Hidronefrosis/genética , Riñón/embriología , Riñón/metabolismo , Mesodermo/metabolismo , Ratones , Ratones Transgénicos , Desarrollo de Músculos/genética , Músculo Liso/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Uréter/metabolismo , Urotelio/embriología , Urotelio/metabolismo
16.
Blood ; 117(17): 4580-9, 2011 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-21364187

RESUMEN

The final stages of of megakaryocyte (MK) maturation involve a series of steps, including polyploidization and proplatelet formation. Although these processes are highly dependent on dynamic changes in the microtubule (MT) cytoskeleton, the mechanisms responsible for regulation of MTs in MKs remain poorly defined. Stathmin is a highly conserved MT-regulatory protein that has been suggested to play a role in MK differentiation of human leukemic cell lines. However, previous studies defining this relationship have reached contradictory conclusions. In this study, we addressed this controversy and investigated the role of stathmin in primary human MKs. To explore the importance of stathmin down-regulation during megakaryocytopoiesis, we used a lentiviral-mediated gene delivery system to prevent physiologic down-regulation of stathmin in primary MKs. We demonstrated that sustained expression of constitutively active stathmin delayed cytoplasmic maturation (ie, glycoprotein GPIb and platelet factor 4 expression) and reduced the ability of MKs to achieve high levels of ploidy. Moreover, platelet production was impaired in MKs in which down-regulation of stathmin expression was prevented. These studies indicate that suppression of stathmin is biologically important for MK maturation and platelet production and support the importance of MT regulation during the final stages of thrombopoiesis.


Asunto(s)
Plaquetas/citología , Megacariocitos/fisiología , Estatmina/genética , Trombopoyesis/fisiología , Células Cultivadas , Citoplasma/fisiología , Regulación hacia Abajo/fisiología , Células HEK293 , Humanos , Lentivirus/genética , Megacariocitos/citología , Microtúbulos/fisiología , Poliploidía , Estatmina/metabolismo
17.
Laryngoscope ; 2023 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-37975487

RESUMEN

OBJECTIVES: Tracheal transplantation is an ideal option for the reconstruction of long-segment circumferential tracheal defects. Our group performed the first successful vascularized single-staged tracheal transplantation in January 2021. Although a rigid biocompatible structure is necessary for a functioning tracheal replacement, the importance of ciliated epithelium, which allows for critical mucociliary clearance, is now being appreciated. Here, we examined the histological changes of the first single-staged human tracheal transplant from serial endoscopic biopsies. METHODS: Biopsies of the tracheal mucosa were serially obtained since the time of the tracheal transplantation. Samples were examined via hematoxylin and eosin, electron microscopy, and immunohistochemistry. RESULTS: One week after transplantation, there is loss of ciliated epithelium and seromucinous cells, with only a basal layer of epithelium remaining. By 2 weeks, however, the epithelium begins to recover, albeit differently depending on the location of the biopsy. Near the site of tracheal anastomosis, there is epithelial proliferation, with the appearance of early ciliated cells. However, in the midgraft, there appears to be evidence of squamous metaplasia. Over time, however, normal ciliated epithelium and mucous cells appear without signs of chronic inflammation. CONCLUSIONS: Critically, the tracheal allograft regained normal appearing respiratory epithelium after initial ischemic injury. The histologic differences at the midgraft versus anastomosis may suggest unique mechanisms of reepithelialization. At the recipient-donor interface, there may be a faster direct migration of recipient-derived epithelial cells, in line with preclinical studies. The midgraft, in contrast, responds with epithelial proliferation from the donor basal cells or dedifferentiated mucous cells. LEVEL OF EVIDENCE: N/A Laryngoscope, 2023.

18.
JCI Insight ; 8(7)2023 04 10.
Artículo en Inglés | MEDLINE | ID: mdl-36853804

RESUMEN

Despite recent progress in the identification of mediators of podocyte injury, mechanisms underlying podocyte loss remain poorly understood, and cell-specific therapy is lacking. We previously reported that kidney and brain expressed protein (KIBRA), encoded by WWC1, promotes podocyte injury in vitro through activation of the Hippo signaling pathway. KIBRA expression is increased in the glomeruli of patients with focal segmental glomerulosclerosis, and KIBRA depletion in vivo is protective against acute podocyte injury. Here, we tested the consequences of transgenic podocyte-specific WWC1 expression in immortalized human podocytes and in mice, and we explored the association between glomerular WWC1 expression and glomerular disease progression. We found that KIBRA overexpression in immortalized human podocytes promoted cytoplasmic localization of Yes-associated protein (YAP), induced actin cytoskeletal reorganization, and altered focal adhesion expression and morphology. WWC1-transgenic (KIBRA-overexpressing) mice were more susceptible to acute and chronic glomerular injury, with evidence of YAP inhibition in vivo. Of clinical relevance, glomerular WWC1 expression negatively correlated with renal survival among patients with primary glomerular diseases. These findings highlight the importance of KIBRA/YAP signaling to the regulation of podocyte structural integrity and identify KIBRA-mediated injury as a potential target for podocyte-specific therapy in glomerular disease.


Asunto(s)
Enfermedades Renales , Podocitos , Humanos , Ratones , Animales , Podocitos/metabolismo , Regulación hacia Arriba , Glomérulos Renales/metabolismo , Transducción de Señal , Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Progresión de la Enfermedad , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo
19.
Cancer Res Commun ; 2(6): 518-532, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35911788

RESUMEN

During the 9/11 attacks individuals were exposed to World Trade Center (WTC) dust which contained a complex mixture of carcinogens. Epidemiological studies have revealed the increased incidence of prostate and thyroid cancer in WTC survivors and responders. While reports have shown that WTC-dust associates with the increased prevalence of inflammatory related disorders, studies to date have not determined whether this exposure impacts cancer progression. In this study, we have used genetically engineered mouse (GEM) models with prostate specific deletion of the PTEN tumor suppressor to study the impact of WTC-dust exposure on deposition of dust particles, inflammation, and cancer progression. In normal C57/BL6 mice, dust exposure increased cellular expression of inflammatory genes with highest levels in the lung and peripheral blood. In normal and tumor bearing GEM mice, increased immune cell infiltration to the lungs was observed. Pathological evaluation of mice at different time points showed that WTC-dust exposure promoted PI3K-AKT activation, increased epithelial proliferation and acinar invasion in prostates with heterozygous and homozygous Pten loss. Using autochthonous and transplant GEM models of prostate cancer we demonstrated that dust exposure caused reduced survival as compared to control cohorts. Finally, we used imaging mass cytometry (IMC) to detect elevated immune cell infiltration and cellular expression of inflammatory markers in prostate tumors isolated from human WTC survivors. Collectively, our study shows that chronic inflammation, induced by WTC dust exposure, promotes more aggressive cancer in genetically predisposed prostates and potentially in patients.


Asunto(s)
Enfermedades Pulmonares , Neoplasias de la Próstata , Animales , Humanos , Masculino , Ratones , Polvo , Inflamación , Fosfatidilinositol 3-Quinasas , Próstata , Neoplasias de la Próstata/epidemiología , Fosfohidrolasa PTEN/genética
20.
Bioconjug Chem ; 22(3): 353-61, 2011 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-21361312

RESUMEN

There are many liver diseases that could be treated with delivery of therapeutics such as DNA, proteins, or small molecules. Nanoparticles are often proposed as delivery vectors for such therapeutics; however, achieving nanoparticle accumulations in the therapeutically relevant hepatocytes is challenging. In order to address this issue, we have synthesized polymer coated, fluorescent iron oxide nanoparticles that bind and deliver DNA, as well as produce contrast for magnetic resonance imaging (MRI), fluorescence imaging, and transmission electron microscopy (TEM). The composition of the coating can be varied in a facile manner to increase the quantity of poly(ethylene glycol) (PEG) from 0% to 5%, 10%, or 25%, with the aim of reducing opsonization but maintaining DNA binding. We investigated the effect of the nanoparticle coating on DNA binding, cell uptake, cell transfection, and opsonization in vitro. Furthermore, we exploited MRI, fluorescence imaging, and TEM to investigate the distribution of the different formulations in the liver of mice. While MRI and fluorescence imaging showed that each formulation was heavily taken up in the liver at 24 h, the 10% PEG formulation was taken up by the therapeutically relevant hepatocytes more extensively than either the 0% PEG or the 5% PEG, indicating its potential for delivery of therapeutics to the liver.


Asunto(s)
Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Hígado/citología , Hígado/metabolismo , Nanopartículas/química , Animales , Transporte Biológico , Supervivencia Celular/efectos de los fármacos , ADN/metabolismo , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/toxicidad , Compuestos Férricos/química , Compuestos Férricos/metabolismo , Compuestos Férricos/farmacocinética , Compuestos Férricos/toxicidad , Células HEK293 , Semivida , Humanos , Imagen por Resonancia Magnética , Ratones , Microscopía Electrónica de Transmisión , Nanopartículas/toxicidad , Polietilenglicoles/química
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