RESUMO
Endothelial cell (EC) dysfunction and atherosclerotic plaque formation coincide with human circulatory regions where blood flow is altered (disturbed). In areas of undisturbed uniform blood flow, including the majority of the vasculature, the vessel wall is relatively atherosclerotic lesion-resistant with normal endothelium. The molecular mechanisms of blood flow regulation of EC function and atherogenesis are unclear. We hypothesize that EC dysfunction potentiating atherosclerosis is related to disturbed flow (DF)-induced EC gap junctional intercellular communication (GJIC) changes via the gap junction connexin (Cx) 37, 40, and 43 proteins, which are involved in EC proliferation and vasoactivity that are known to be altered in atherosclerosis. We investigated human EC GJIC using an in vitro model of the hemodynamic features found in atherosclerotic-prone DF regions in vivo. Using dye transfer assays, Cx-specific mimetic peptide inhibitors, proliferation assays, and immunocytochemistry, we correlated functional GJIC via gap junction channels formed by hemichannels composed of the two most abundant endothelial Cx-Cx40 and Cx43-to EC proliferation and expression of vasoactive endothelial-type nitric oxide synthase (eNOS). We found that, in uniform flow conditions, substantial GJIC was conducted through gap junctions containing Cx40 hemichannels and correlated to a nonproliferative EC phenotype and membrane localization of eNOS, similar to physiological conditions. In DF, GJIC was largely attained through Cx43 hemichannel-containing gap junctions, EC phenotype was proliferative (attributed to loss of contact inhibition), and intracellular eNOS was more abundant than membrane eNOS, typical of atherosclerotic sites in vivo. This is the first in vitro study to demonstrate local hemodynamically defined Cx protein specificity in human EC GJIC with a potential role in endothelial dysfunction characteristic of early atherosclerosis.
Assuntos
Comunicação Celular , Conexinas/metabolismo , Células Endoteliais/metabolismo , Junções Comunicantes/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Proliferação de Células , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Humanos , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse MecânicoRESUMO
Within the cellular microenvironment, extracellular matrix (ECM) proteins are critical nonsoluble signaling factors that modulate cell attachment, migration, proliferation, and differentiation. We have developed a simple method to isolate and process ECM from endothelial cell cultures to create a three-dimensional (3D) ECM substrate. Endothelial cell monolayers were chemically lysed and enzymatically digested to isolate a thin, two-dimensional (2D) ECM substrate. This thin 1.8 µm 2D ECM was collected and applied to a solid support to produce 12-16-fold thicker 3D ECM substrates with average thicknesses ranging from 21 to 29 µm. The biological activity of isolated ECM was assessed by cell culture. Neural progenitor cells were cultured on endothelial-produced ECM, and unlike the thin 2D ECM, which was quickly remodeled by cells, 3D ECM substrates remained in culture for an extended period (>7 days), suggesting that a continuous signaling cue for in vitro experiments may be provided. This simple method for creating 3D ECM substrates can be applied to a variety of cell culture models for studies aimed at identifying the signaling effects of the ECM within cellular microenvironments.
Assuntos
Técnicas de Cultura de Células/métodos , Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Animais , Células Cultivadas , Centrifugação , Camundongos , Células-Tronco Neurais/citologiaRESUMO
Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.
Assuntos
Aorta/fisiologia , Velocidade do Fluxo Sanguíneo/fisiologia , Comunicação Celular/fisiologia , Conexinas/metabolismo , Células Endoteliais/fisiologia , Junções Comunicantes/metabolismo , Aorta/citologia , Células Cultivadas , Humanos , Ligação ProteicaRESUMO
Regulation of endothelial cell (EC) permeability by bioactive molecules is associated with specific patterns of cytoskeletal and cell contact remodeling. A role for mechanical factors such as shear stress (SS) and cyclic stretch (CS) in cytoskeletal rearrangements and regulation of EC permeability becomes increasingly recognized. This paper examined redistribution of focal adhesion (FA) proteins, site-specific focal adhesion kinase (FAK) phosphorylation, small GTPase activation and barrier regulation in human pulmonary EC exposed to laminar shear stress (15 dyn/cm2) or cyclic stretch (18% elongation) in vitro. SS caused peripheral accumulation of FAs, whereas CS induced randomly distributed FAs attached to the ends of newly formed stress fibers. SS activated small GTPase Rac without effects on Rho, whereas 18% CS activated without effect on Rac. SS increased transendothelial electrical resistance (TER) in EC monolayers, which was further elevated by barrier-protective phospholipid sphingosine 1-phosphate. Finally, SS induced FAK phosphorylation at Y576, whereas CS induced FAK phosphorylation at Y397 and Y576. These results demonstrate for the first time differential effects of SS and CS on Rho and Rac activation, FA redistribution, site-specific FAK phosphorylation, and link them with SS-mediated barrier enhancement. Thus, our results suggest common signaling and cytoskeletal mechanisms shared by mechanical and chemical factors involved in EC barrier regulation.
Assuntos
Adesões Focais/enzimologia , Pulmão/citologia , Pulmão/enzimologia , Proteínas Tirosina Quinases/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular/análise , Proteínas do Citoesqueleto/análise , Elasticidade , Células Endoteliais/química , Células Endoteliais/enzimologia , Células Endoteliais/ultraestrutura , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Adesões Focais/ultraestrutura , Proteínas Ativadoras de GTPase/análise , Humanos , Paxilina , Fosfoproteínas/análise , Fosforilação , Estresse MecânicoRESUMO
Endothelial sequestration of circulating monocytes is a key event in early atherosclerosis. Hemodynamics is proposed to regulate monocyte-endothelial cell interactions by direct cell activation and establishment of flow environments that are conducive or prohibitive to cell-cell interaction. We investigated fluid shear regulation of monocyte-endothelial cell adhesion in vitro using a disturbed laminar shear system that models in vivo hemodynamics characteristic of lesion-prone vascular regions. Human endothelial cell monolayers were flow conditioned for 6 h before evaluation of monocyte adhesion under static and dynamic flow conditions. Results revealed a distinctive clustered cell pattern of monocyte adhesion that strongly resembles in vivo leukocyte adhesion in early- and late-stage atherosclerosis. Clustered monocyte cell adhesion correlated with endothelial cells coexpressing intercellular adhesion molecule-1 (ICAM-1) and E-selectin as result of a flow-induced, selective upregulation of E-selectin expression in a subset of ICAM-1-expressing cells. Clustered monocyte cell adhesion assayed under static conditions exhibited a spatial variation in size and frequency of occurrence, which demonstrates differential regulation of endothelial cell adhesiveness by the local flow environment. Dynamic adhesion studies conducted with circulating monocytes resulted in clustered cell adhesion only within the disturbed flow region, where the monocyte rate of motion is sufficiently low for cell-cell interaction. These studies provide evidence and reveal mechanisms of local hemodynamic regulation of endothelial adhesiveness and endothelial monocyte interaction that lead to localized monocyte adhesion and potentially contribute to the focal origin of arterial diseases such as atherosclerosis.
Assuntos
Selectina E/metabolismo , Células Endoteliais/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/fisiologia , Adesão Celular/fisiologia , Agregação Celular/fisiologia , Células Cultivadas , Humanos , Estresse Mecânico , Veias Umbilicais/citologiaRESUMO
The luminal surface of rat lung microvascular endothelial cells in situ is sensitive to changing hemodynamic parameters. Acute mechanosignaling events initiated in response to flow changes in perfused lung microvessels are localized within specialized invaginated microdomains called caveolae. Here we report that chronic exposure to shear stress alters caveolin expression and distribution, increases caveolae density, and leads to enhanced mechanosensitivity to subsequent changes in hemodynamic forces within cultured endothelial cells. Flow-preconditioned cells expressed a fivefold increase in caveolin (and other caveolar-residing proteins) at the luminal surface compared with no-flow controls. The density of morphologically identifiable caveolae was enhanced sixfold at the luminal cell surface of flow-conditioned cells. Laminar shear stress applied to static endothelial cultures (flow step of 5 dyn/cm2), enhanced the tyrosine phosphorylation of luminal surface proteins by 1.7-fold, including caveolin-1 by 1.3-fold, increased Ser1179 phosphorylation of endothelial nitric oxide synthase (eNOS) by 2.6-fold, and induced a 1.4-fold activation of mitogen-activated protein kinases (ERK1/2) over no-flow controls. The same shear step applied to endothelial cells preconditioned under 10 dyn/cm2 of laminar shear stress for 6 h and induced a sevenfold increase of total phosphotyrosine signal at the luminal endothelial cell surface enhanced caveolin-1 tyrosine phosphorylation 5.8-fold and eNOS phosphorylation by 3.3-fold over static control values. In addition, phosphorylated caveolin-1 and eNOS proteins were preferentially localized to caveolar microdomains. In contrast, ERK1/2 activation was not detected in conditioned cells after acute shear challenge. These data suggest that cultured endothelial cells respond to a sustained flow environment by directing caveolae to the cell surface where they serve to mediate, at least in part, mechanotransduction responses.
Assuntos
Cavéolas/fisiologia , Condicionamento Psicológico , Endotélio Vascular/fisiologia , Mecanorreceptores/fisiologia , Circulação Pulmonar/fisiologia , Transdução de Sinais/fisiologia , Animais , Bovinos , Cavéolas/ultraestrutura , Caveolina 1 , Caveolinas/metabolismo , Membrana Celular/fisiologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Técnicas In Vitro , Microscopia Eletrônica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas/metabolismo , Estresse Mecânico , Fatores de Tempo , Distribuição TecidualRESUMO
The novel hydrostrain system was designed in an effort to establish and maintain conditions that simulate the in-vivo mechanical environment of the bladder. In this laboratory system, ovine bladder smooth muscle cells on flexible, 10-cm-dia silastic membranes were exposed simultaneously to hydrostatic pressure (40 cm H2O, a pressure level currently associated with bladder pathologies) and mechanical strains (up to 25 percent) under standard cell culture conditions for 7 h. Under these conditions, Heparin Binding-Epidermal Growth Factor and Collagen Type III mRNA expression were significantly increased (p<0.01 and 0.1, respectively); however, no changes were observed in Collagen Type I mRNA expression. Decreases in the Collagen Type I:Type III ratio following simultaneous exposure of bladder smooth muscle cells to pathological levels of hydrostatic pressure and mechanical strain in vitro are in agreement with clinically observed increases in Collagen Type III with concomitant decreased human bladder compliance. The results of the present study, therefore, provide cellular/molecular level information relevant to bladder pathology that could have significant implications in the field of clinical urology.
Assuntos
Regulação da Expressão Gênica/fisiologia , Membranas Artificiais , Miócitos de Músculo Liso/fisiologia , Reologia/instrumentação , Bexiga Urinária/fisiologia , Animais , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo III/genética , Dimetilpolisiloxanos/química , Fator de Crescimento Epidérmico/genética , Desenho de Equipamento , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Pressão Hidrostática , Peptídeos e Proteínas de Sinalização Intercelular , Miócitos de Músculo Liso/citologia , Valores de Referência , Reologia/métodos , Ovinos , Silicones/química , Estresse Mecânico , Bexiga Urinária/citologia , Bexiga Urinária/embriologiaRESUMO
Hemodynamic forces in the form of shear stress (SS) and mechanical strain imposed by circulating blood are recognized factors involved in the control of systemic endothelial cell (EC) cytoskeletal structure and function. However, the effects of acute SS on pulmonary endothelium have not been precisely characterized, nor the mechanism of rapid SS-induced EC cytoskeletal rearrangement understood. We exposed bovine and human pulmonary EC monolayers to laminar SS (10 dynes/cm2) in a parallel plate flow chamber and observed increased actin stress fiber formation 15 min after application of flow. Acute SS-induced pronounced cortical cytoskeletal rearrangement characterized by myosin light chain kinase (MLCK)- and Rho-associated kinase (RhoK)-dependent accumulation of diphosphorylated regulatory myosin light chains (MLC) in the cortical actin ring, junctional protein tyrosine phosphorylation, and transient peripheral translocation of cortactin, an actin-binding protein involved in the regulation of actin polymerization. SS-induced cortactin translocation was independent of Erk-1,2 MAP kinase, p60(Src), MLCK, or RhoK activities, and unaffected by overexpression of a cortactin mutant lacking four major p60(Src) phosphorylation sites. However, both SS-induced transient cortactin translocation and cytoskeletal reorientation in response to sustained (24 h) SS was abolished in cells overexpressing either dominant negative Rac 1 or a dominant negative construct of its downstream target, p21-activated kinase (PAK)-1. Our results suggest a potential role for cortactin in the SS-induced EC cortical cytoskeletal remodeling and demonstrate a novel mechanism of Rac GTPase-dependent regulation of the pulmonary endothelial cytoskeleton by SS.