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1.
Elife ; 42015 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-25643397

RESUMO

Vascular remodeling under conditions of growth or exercise, or during recovery from arterial restriction or blockage is essential for health, but mechanisms are poorly understood. It has been proposed that endothelial cells have a preferred level of fluid shear stress, or 'set point', that determines remodeling. We show that human umbilical vein endothelial cells respond optimally within a range of fluid shear stress that approximate physiological shear. Lymphatic endothelial cells, which experience much lower flow in vivo, show similar effects but at lower value of shear stress. VEGFR3 levels, a component of a junctional mechanosensory complex, mediate these differences. Experiments in mice and zebrafish demonstrate that changing levels of VEGFR3/Flt4 modulates aortic lumen diameter consistent with flow-dependent remodeling. These data provide direct evidence for a fluid shear stress set point, identify a mechanism for varying the set point, and demonstrate its relevance to vessel remodeling in vivo.


Assuntos
Estresse Fisiológico , Veias Umbilicais/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Remodelação Vascular , Animais , Endotélio Vascular/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Peixe-Zebra
2.
Proc Natl Acad Sci U S A ; 111(48): 17308-13, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25404299

RESUMO

Atherosclerotic plaque localization correlates with regions of disturbed flow in which endothelial cells (ECs) align poorly, whereas sustained laminar flow correlates with cell alignment in the direction of flow and resistance to atherosclerosis. We now report that in hypercholesterolemic mice, deletion of syndecan 4 (S4(-/-)) drastically increased atherosclerotic plaque burden with the appearance of plaque in normally resistant locations. Strikingly, ECs from the thoracic aortas of S4(-/-) mice were poorly aligned in the direction of the flow. Depletion of S4 in human umbilical vein endothelial cells (HUVECs) using shRNA also inhibited flow-induced alignment in vitro, which was rescued by re-expression of S4. This effect was highly specific, as flow activation of VEGF receptor 2 and NF-κB was normal. S4-depleted ECs aligned in cyclic stretch and even elongated under flow, although nondirectionally. EC alignment was previously found to have a causal role in modulating activation of inflammatory versus antiinflammatory pathways by flow. Consistent with these results, S4-depleted HUVECs in long-term laminar flow showed increased activation of proinflammatory NF-κB and decreased induction of antiinflammatory kruppel-like factor (KLF) 2 and KLF4. Thus, S4 plays a critical role in sensing flow direction to promote cell alignment and inhibit atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais , Sindecana-4/metabolismo , Animais , Aterosclerose/genética , Western Blotting , Células Cultivadas , Células Endoteliais/citologia , Feminino , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , NF-kappa B/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Mecânico , Sindecana-4/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
3.
PLoS One ; 8(9): e73389, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24039928

RESUMO

Adhesions are multi-molecular complexes that transmit forces generated by a cell's acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions' mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement.


Assuntos
Citoesqueleto de Actina/metabolismo , Aplysia/citologia , Moléculas de Adesão Celular/metabolismo , Animais , Aplysia/metabolismo , Adesão Celular , Células Cultivadas
4.
Cell ; 148(1-2): 175-88, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22265410

RESUMO

Little is known about how neutrophils and other cells establish a single zone of actin assembly during migration. A widespread assumption is that the leading edge prevents formation of additional fronts by generating long-range diffusible inhibitors or by sequestering essential polarity components. We use morphological perturbations, cell-severing experiments, and computational simulations to show that diffusion-based mechanisms are not sufficient for long-range inhibition by the pseudopod. Instead, plasma membrane tension could serve as a long-range inhibitor in neutrophils. We find that membrane tension doubles during leading-edge protrusion, and increasing tension is sufficient for long-range inhibition of actin assembly and Rac activation. Furthermore, reducing membrane tension causes uniform actin assembly. We suggest that tension, rather than diffusible molecules generated or sequestered at the leading edge, is the dominant source of long-range inhibition that constrains the spread of the existing front and prevents the formation of secondary fronts.


Assuntos
Quimiotaxia de Leucócito , Neutrófilos/citologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Polaridade Celular , Humanos , Neutrófilos/metabolismo , Pseudópodes/metabolismo
5.
Nat Methods ; 6(12): 905-9, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19915561

RESUMO

Molecular gradients are important for various biological processes including the polarization of tissues and cells during embryogenesis and chemotaxis. Investigations of these phenomena require control over the chemical microenvironment of cells. We present a technique to set up molecular concentration patterns that are chemically, spatially and temporally flexible. Our strategy uses optically manipulated microsources, which steadily release molecules. Our technique enables the control of molecular concentrations over length scales down to about 1 microm and timescales from fractions of a second to an hour. We demonstrate this technique by manipulating the motility of single human neutrophils. We induced directed cell polarization and migration with microsources loaded with the chemoattractant formyl-methionine-leucine-phenylalanine. Furthermore, we triggered highly localized retraction of lamellipodia and redirection of polarization and migration with microsources releasing cytochalasin D, an inhibitor of actin polymerization.


Assuntos
Neutrófilos/citologia , Óptica e Fotônica , Movimento Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos
6.
Opt Express ; 17(8): 6209-17, 2009 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-19365444

RESUMO

We describe open-loop and closed-loop multiplexed force measurements using holographic optical tweezers. We quantify the performance of our novel video-based control system in a driven suspension of colloidal particles. We demonstrate our system's abilities with the measurement of the mechanical coupling between Aplysia bag cell growth cones and beads functionalized with the neuronal cell adhesion molecule, apCAM. We show that cells form linkages which couple beads to the underlying cytoskeleton. These linkages are intermittent, stochastic and heterogeneous across beads distributed near the leading edge of a single growth cone.


Assuntos
Aplysia/citologia , Aplysia/fisiologia , Cones de Crescimento/fisiologia , Cones de Crescimento/ultraestrutura , Holografia/instrumentação , Micromanipulação/instrumentação , Pinças Ópticas , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estresse Mecânico
7.
Langmuir ; 24(4): 1160-4, 2008 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-18062711

RESUMO

We study the electrostatic and hydrodynamic interactions of colloidal particles in nonpolar solvents. Using blinking optical tweezers, we can extract the screening length, kappa-1, the effective surface potential, |ezeta*|, and the hydrodynamic radius, ah, in a single measurement. We apply this technique to suspensions of polystyrene and poly(methyl methacrylate) particles in hexadecane with soluble charge control agents, aerosol sodium di-2-ethylhexylsulfosuccinate (AOT) and polyisobutylene succinimide (OLOA-1200). We find that the electrostatic interactions of these particles depend sensitively on surface composition as well as on the concentration and chemistry of the charge control agent.


Assuntos
Alcanos/química , Coloides/química , Polímeros/química , Polimetil Metacrilato/química , Poliestirenos/química , Succinatos/química , Succinimidas/química , Aerossóis/química , Pinças Ópticas , Tamanho da Partícula , Solventes/química , Eletricidade Estática , Propriedades de Superfície , Termodinâmica
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