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1.
Australas J Ultrasound Med ; 26(2): 85-90, 2023 May.
Artículo en Inglés | MEDLINE | ID: mdl-37252625

RESUMEN

Introduction/Purpose: Measurement of jugular venous pressure (JVP) by novice clinicians can be unreliable, particularly when evaluating obese patients. Measurement of JVP using ultrasound (uJVP) is simple to perform and provides accurate measurements. This study evaluated whether students and residents inexperienced with ultrasound could rapidly be taught to measure JVP using ultrasound in obese patients with the same accuracy as cardiologists measuring JVP via physical examination. Additionally, this study also evaluated the correlation between qualitative and quantitative JVP assessment. Methods: This prospective, blinded study compared uJVP measurements performed by novice clinicians after brief training to JVP measurements performed by cardiologists (cJVP) on physical examination. Association between uJVP and cJVP was assessed using linear correlation, agreement and bias were assessed using the Bland-Altman analysis and inter-rater reliability of uJVP was assessed using intraclass correlation coefficient (ICC). The association between qualitative and quantitative JVP assessment was assessed using linear correlation. Results: Novice clinicians (n = 16) obtained 34 measurements from 26 patients (mean BMI 35.5) and reported moderate-to-high confidence in all measurements. uJVP correlated well with cJVP (r = 0.73) with an average error of 0.06 cm. The estimated uJVP ICC was 0.83 (95% CI = 0.44, 0.96). Qualitative uJVP had only a moderate correlation (r = 0.63) to quantitative uJVP. Discussion: Novice clinicians often have difficulty assessing JVP on physical examination, particularly in obese patients. Our findings show a high degree of correlation between JVP measurements performed by novice clinicians using ultrasound compared with JVP measurements made by experienced cardiologists on physical examination. Furthermore, novice clinicians were able to be trained quickly, their measurements were determined to be accurate and precise and they expressed moderate-to-high confidence in their results. Conclusions: After brief training, novice clinicians were able to accurately assess JVP in obese patients as compared to measurements made by experienced cardiologists on physical examination. Results suggest that ultrasound may greatly improve novice clinicians' JVP assessment accuracy, particularly in obese patients.

2.
PLoS One ; 7(3): e33915, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22470492

RESUMEN

Myofibroblasts (Mfs) that persist in a healing wound promote extracellular matrix (ECM) accumulation and excessive tissue contraction. Increased levels of integrin αvß5 promote the Mf phenotype and other fibrotic markers. Previously we reported that maintaining uPA (urokinase plasminogen activator) bound to its cell-surface receptor, uPAR prevented TGFß-induced Mf differentiation. We now demonstrate that uPA/uPAR controls integrin ß5 protein levels and in turn, the Mf phenotype. When cell-surface uPA was increased, integrin ß5 levels were reduced (61%). In contrast, when uPA/uPAR was silenced, integrin ß5 total and cell-surface levels were increased (2-4 fold). Integrin ß5 accumulation resulted from a significant decrease in ß5 ubiquitination leading to a decrease in the degradation rate of internalized ß5. uPA-silencing also induced α-SMA stress fiber organization in cells that were seeded on collagen, increased cell area (1.7 fold), and increased integrin ß1 binding to the collagen matrix, with reduced activation of ß1. Elevated cell-surface integrin ß5 was necessary for these changes after uPA-silencing since blocking αvß5 function reversed these effects. Our data support a novel mechanism by which downregulation of uPA/uPAR results in increased integrin αvß5 cell-surface protein levels that regulate the activity of ß1 integrins, promoting characteristics of the persistent Mf.


Asunto(s)
Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Receptores de Vitronectina/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Actinas/metabolismo , Anticuerpos/inmunología , Células Cultivadas , Colágeno/metabolismo , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Miofibroblastos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Receptores de Vitronectina/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitinación , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/genética
3.
Mol Vis ; 17: 2835-46, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22128231

RESUMEN

PURPOSE: There is an unmet challenge to promote wound healing in non-healing wounds such as in the post-LASIK (laser-assisted in situ keratomileusis) cornea. Using human corneal fibroblasts (HCFs) in cell culture, we investigated the concentration dependence of the growth factor transforming growth factor ß1 (TGFß1) on wound closure. Although high concentrations of TGFß1 leads to scarring, we asked whether low concentrations of TGFß1 could promote wound healing without generating a large fibrotic response. METHODS: HCFs were cultured in supplemented serum-free media (SSFM). Cell migration was assessed by scratch-wounding. SMAD 2/3 and p38 mitogen-activated protein kinase (p38MAPK) localization and α-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry. Active TGFß was quantified using a luciferase bio-assay. RESULTS: We found that neutralizing antibody to TGFß1 reduced cell migration by 73%, compared to immunoglobulin G (IgG) control, establishing that endogenous TGFß1 (determined to be 0.01 ng/ml) is necessary to promote cell migration. To evaluate the concentration-dependent effects of TGFß1 on wound closure, HCF migration was quantified to determine the impact of increasing concentrations of TGFß1 (0.01-1.0 ng/ml). Compared to control (cells in SSFM), the higher concentrations (0.1 and 1.0 ng/ml TGFß1) significantly decreased cell migration (63%-86%), induced myofibroblast differentiation (83%-88%), increased SMAD 2/3 localization into the nucleus (72%-79%) and inhibited the activation of p38MAPK (51%-63%). In contrast, addition of the lower concentration of TGFß1 (0.01 ng/ml TGFß1) promoted a cell migration rate that was similar to endogenous TGFß, reduced SMAD 2/3 nuclear localization, and stimulated p38MAPK activation. A TGFß1 blocking antibody and the p38MAPK inhibitor, SB202192, was used to demonstrate that p38MAPK activation is necessary for TGFß1-induced cell migration. CONCLUSIONS: Together, our data demonstrate that low concentrations of TGFß1 promote p38MAPK activation that is a key to HCF migration, suggesting that a low concentration of TGFß may be useful in treating non-healing corneal wounds.


Asunto(s)
Movimiento Celular/efectos de los fármacos , Córnea/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Cicatrización de Heridas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Actinas/genética , Actinas/metabolismo , Anticuerpos Bloqueadores/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Córnea/citología , Córnea/metabolismo , Córnea/cirugía , Medio de Cultivo Libre de Suero , Relación Dosis-Respuesta a Droga , Activación Enzimática , Fibroblastos/citología , Humanos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/fisiología , Proteínas Smad/genética , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genética
4.
J Biol Chem ; 284(31): 20708-17, 2009 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-19487690

RESUMEN

Fibrosis is characterized by elevated transforming growth factor beta (TGFbeta) signaling, resulting in extracellular matrix accumulation and increased PAI-1 (plasminogen activator inhibitor) expression. PAI-1 induces the internalization of urokinase plasminogen activator/receptor and integrin alphavbeta3 from the cell surface. Since increased alphavbeta3 expression correlates with increased TGFbeta signaling, we hypothesized that aberrant PAI-1-mediated alphavbeta3 endocytosis could initiate an autocrine loop of TGFbeta activity. We found that in PAI-1 knock-out (KO) mouse embryonic fibroblasts), alphavbeta3 endocytosis was reduced by approximately 75%, leaving alphavbeta3 in enlarged focal adhesions, similar to wild type cells transfected with PAI-1 small interfering RNA. TGFbeta signaling was significantly enhanced in PAI-1 KO cells, as demonstrated by a 3-fold increase in SMAD2/3-containing nuclei and a 2.9-fold increase in TGFbeta activity that correlated with an increase in alphavbeta3 and TGFbeta receptor II expression. As expected, PAI-1 KO cells had unregulated plasmin activity, which was only partially responsible for TGFbeta activation, as evidenced by a mere 25% reduction in TGFbeta activity when plasmin was inhibited. Treatment of cells with an alphavbeta3-specific cyclic RGD peptide (GpenGRGD) led to a more profound (59%) TGFbeta inhibition; a nonspecific RGD peptide (GRGDNP) inhibited TGFbeta by only 23%. Human primary fibroblasts were used to confirm that PAI-1 inhibition and beta3 overexpression led to an increase in TGFbeta activity. Consistent with a fibrotic phenotype, PAI-1 KO cells were constitutively myofibroblasts that had a 1.6-fold increase in collagen deposition over wild type cells. These data suggest that PAI-1-mediated regulation of alphavbeta3 integrin is critical for the control of TGFbeta signaling and the prevention of fibrotic disease.


Asunto(s)
Comunicación Autocrina , Integrina alfaVbeta3/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Colágeno/biosíntesis , ADN Complementario/metabolismo , Endocitosis , Fibrinolisina/metabolismo , Fibroblastos/metabolismo , Adhesiones Focales/metabolismo , Humanos , Ratones , Ratones Noqueados , Visón , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo
5.
Mol Cell ; 32(2): 169-79, 2008 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-18951085

RESUMEN

Elevated catecholamines in the heart evoke transcriptional activation of the Myocyte Enhancer Factor (MEF) pathway to induce a cellular response known as pathological myocardial hypertrophy. We have discovered that the A-Kinase Anchoring Protein (AKAP)-Lbc is upregulated in hypertrophic cardiomyocytes. It coordinates activation and movement of signaling proteins that initiate MEF2-mediated transcriptional reprogramming events. Live-cell imaging, fluorescent kinase activity reporters, and RNA interference techniques show that AKAP-Lbc couples activation of protein kinase D (PKD) with the phosphorylation-dependent nuclear export of the class II histone deacetylase HDAC5. These studies uncover a role for AKAP-Lbc in which increased expression of the anchoring protein selectively amplifies a signaling pathway that drives cardiac myocytes toward a pathophysiological outcome.


Asunto(s)
Proteínas de Anclaje a la Quinasa A/fisiología , Cardiomegalia/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal , Proteínas 14-3-3/metabolismo , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Transporte Activo de Núcleo Celular , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ventrículos Cardíacos/efectos de los fármacos , Histona Desacetilasas/metabolismo , Humanos , Factores de Transcripción MEF2 , Antígenos de Histocompatibilidad Menor , Modelos Biológicos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Factores Reguladores Miogénicos/metabolismo , Fenilefrina/farmacología , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Ratas
6.
J Biol Chem ; 282(45): 33155-67, 2007 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-17827149

RESUMEN

The movement of signal transduction enzymes in and out of multi-protein complexes coordinates the spatial and temporal resolution of cellular events. Anchoring and scaffolding proteins are key to this process because they sequester protein kinases and phosphatases with a subset of their preferred substrates. The protein kinase A-anchoring family of proteins (AKAPs), which target the cAMP-dependent protein kinase (PKA) and other enzymes to defined subcellular microenvironments, represent a well studied group of these signal-organizing molecules. In this report we demonstrate that the Rab27a GTPase effector protein MyRIP is a member of the AKAP family. The zebrafish homolog of MyRIP (Ze-AKAP2) was initially detected in a two-hybrid screen for AKAPs. A combination of biochemical, cell-based, and immunofluorescence approaches demonstrate that the mouse MyRIP ortholog targets the type II PKA holoenzyme via an atypical mechanism to a specific perinuclear region of insulin-secreting cells. Similar approaches show that MyRIP interacts with the Sec6 and Sec8 components of the exocyst complex, an evolutionarily conserved protein unit that controls protein trafficking and exocytosis. These data indicate that MyRIP functions as a scaffolding protein that links PKA to components of the exocytosis machinery.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Anclaje a la Quinasa A/química , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/clasificación , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética
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