RESUMEN

Observations from diverse studies on cell biomechanics and mechanobiology reveal that altered mechanical stimuli can induce significant changes in cytoskeletal organization, focal adhesion complexes, and overall mechanical properties. To investigate effects of short-term equibiaxial stretching on the transverse stiffness of and remodeling of focal adhesions in vascular smooth muscle cells, we developed a cell-stretching device that can be combined with both atomic force and confocal microscopy. Results demonstrate that cyclic 10%, but not 5%, equibiaxial stretching at 0.25 Hz significantly and rapidly alters both cell stiffness and focal adhesion associated paxillin and vinculin. Moreover, measured changes in stiffness and focal adhesion area from baseline values tend to correlate well over the durations of stretching studied. It is suggested that remodeling of focal adhesions plays a critical role in regulating cell stiffness by recruiting and anchoring actin filaments, and that cells rapidly remodel in an attempt to maintain a homeostatic biomechanical state when perturbed above a threshold value.

Asunto(s)

Adhesión Celular/fisiología , Proteínas del Citoesqueleto/fisiología , Citoesqueleto/fisiología , Adhesiones Focales/fisiología , Mecanotransducción Celular/fisiología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/fisiología , Animales , Anisotropía , Células Cultivadas , Simulación por Computador , Elasticidad , Cinética , Modelos Biológicos , Ratas , Estrés Mecánico , Factores de Tiempo

RESUMEN

A constrained mixture theory model was developed and used to estimate remodeling of F-actin in vascular smooth muscle cells that were subjected to 10% equibiaxial stretching for up to 30min. The model was based on a synthesis of data on time-dependent changes in atomic force microscopy measured cell stiffness and immunofluorescence measured focal adhesion associated vinculin as well as data on stress fiber stiffness and pre-stretch. Results suggest that an observed acute (after 2min of stretching) increase in cell stiffness is consistent with an increased stretch of the originally present F-actin plus an assembly of new F-actin having nearly homeostatic values of stretch. Moreover, the subsequent (after 30min of stretching) decrease in cell stiffness back towards the baseline value is consistent with a replacement of the overstretched original filaments with the new (reassembled), less stretched filaments. That is, overall cell response is consistent with a recently proposed concept of "tensional homeostasis" whereby cells seek to maintain constant certain mechanical factors via a remodeling of intracellular and transmembrane proteins. Although there is a need to refine the model based on more comprehensive data sets, using multiple experimental approaches, the present results suggest that a constrained mixture theory can capture salient features of the dynamics of F-actin remodeling and that it offers some advantages over many past methods of modeling, particularly those based on classical linearized viscoelasticity.

Asunto(s)

Actinas/ultraestructura , Citoesqueleto/ultraestructura , Músculo Liso Vascular/ultraestructura , Animales , Células Cultivadas , Elasticidad , Adhesiones Focales/metabolismo , Humanos , Microscopía Fluorescente , Modelos Biológicos , Fibras de Estrés/ultraestructura , Estrés Mecánico , Vinculina/fisiología

RESUMEN

To study the complex interaction between oxidative injury and the pathogenesis of vascular disease, vascular gene expression was examined in male Sprague-Dawley rats given 35 or 70 mg/kg allylamine, a synthetic amine converted to acrolein and hydrogen peroxide within the vascular wall. Vascular lesions and extensive vascular remodeling, coupled to increased production of 8-epi-PGF2alpha, nuclear localization of NFkappaB, and alterations in glutathione homeostasis, were observed in animals treated with allylamine for up to 20 days. Transcriptional profiling, immunohistochemistry, and in situ hybridization showed that genes involved in adhesion and extracellular matrix (ECM) (alpha(1) integrin, collagen), cytoskeletal rearrangements (alpha-smooth muscle actin, alpha-tropomyosin), and signal transduction (NFkappaB, osteopontin, and LINE) were altered by oxidant treatment. To evaluate mechanisms of gene dysregulation, cultured aortic smooth muscle cells were challenged with allylamine or its metabolites and processed for molecular analysis. These agents increased formation of reactive oxygen species and elicited changes in gene expression similar to those observed in vivo. Oxidative stress and changes in gene expression were inhibited by N-acetyl cysteine, a precursor of glutathione. These results indicate that genes along the ECM-integrin-cytoskeletal axis, in addition to LINE, are molecular targets in oxidant-induced vascular injury.

Asunto(s)

Oxidantes/farmacología , Acetilcisteína/metabolismo , Acroleína/metabolismo , Acroleína/farmacología , Alilamina/metabolismo , Alilamina/farmacología , Animales , Aorta/metabolismo , Western Blotting , Análisis por Conglomerados , Citoesqueleto/metabolismo , Dinoprost/análogos & derivados , Dinoprost/biosíntesis , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Genoma , Glutatión/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Procesamiento de Imagen Asistido por Computador , Hibridación Fluorescente in Situ , Integrina alfa1/metabolismo , Integrinas/metabolismo , Masculino , Microscopía Fluorescente , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteopontina , Oxidantes/metabolismo , Estrés Oxidativo , Oxígeno/metabolismo , ARN/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sialoglicoproteínas/metabolismo , Tropomiosina/química , Tropomiosina/metabolismo

RESUMEN

98Emphasis on the individual investigator has fostered discovery for centuries, yet it is now recognized that the complexity of problems in the biomedical sciences and engineering requires collaborative efforts from individuals having diverse training and expertise. Various approaches can facilitate interdisciplinary interactions, but we submit that there is a critical need for a new educational paradigm for the way that we train biomedical engineers, life scientists, and mathematicians. We cannot continue to train graduate students in isolation within single disciplines, nor can we ask any one individual to learn all the essentials of biology, engineering, and mathematics. We must transform how students are trained and incorporate how real-world research and development are done-in diverse, interdisciplinary teams. Our fundamental vision is to create an innovative paradigm for graduate research and training that yields a new generation of biomedical engineers, life scientists, and mathematicians that is more diverse and that embraces and actively pursues a truly interdisciplinary, team-based approach to research based on a known benefit and mutual respect. In this paper, we describe our attempt to accomplish this via focused training in biomechanics, biomedical optics, mathematics, mechanobiology, and physiology. The overall approach is applicable, however, to most areas of biomedical research.

Asunto(s)

Disciplinas de las Ciencias Biológicas/educación , Ingeniería Biomédica/educación , Investigación Biomédica/métodos , Educación de Postgrado/métodos , Disciplinas de las Ciencias Biológicas/tendencias , Ingeniería Biomédica/tendencias , Educación de Postgrado/tendencias , Humanos

RESUMEN

Atomic force microscopy (AFM) is one of many new technologies available to study the mechanical properties and mechanobiological responses of living cells. Despite the widespread usage of this technology, there has been little attempt to develop new theoretical frameworks to interpret the associated data. Rather, most analyses rely on the classical Hertz solution for the indentation of an elastic half-space within the context of linearized elasticity. In contrast, we propose a fully nonlinear, constrained mixture model for adherent cells that allows one to account separately for the contributions of the three primary structural constituents of the cytoskeleton. Moreover, we extend a prior solution for a small indentation superimposed on a finite equibiaxial extension by incorporating in this mixture model for the special case of an initially random distribution of constituents (actin, intermediate filaments, and microtubules). We submit that this theoretical framework will allow an improved interpretation of indentation force-depth data from a sub-class of atomic force microscopy tests and will serve as an important analytical check for future finite element models. The latter will be necessary to exploit further the capabilities of both atomic force microscopy and nonlinear mixture theories for cell behavior.

Asunto(s)

Citoesqueleto/ultraestructura , Microscopía de Fuerza Atómica , Fenómenos Biomecánicos , Adhesión Celular

RESUMEN

Under normoxic conditions in vitro, isolated pulmonary arteries from broilers exhibit reduced endothelium-dependent relaxation responses when compared with Leghorns. In vivo, hypoxia increases the susceptibility of broiler chickens to pulmonary hypertension syndrome (PHS), whereas Leghorns are considered resistant to PHS. Because L-arginine supplementation decreases the incidence of PHS in vivo and improves the relaxation responses of broiler isolated pulmonary arteries in vitro, we hypothesized that in vitro hypoxia would further reduce the relaxation responses of broilers to endothelium-derived nitric oxide (EDNO)-dependent vasodilators and that L-arginine supplementation would alleviate this impairment. As a test of this hypothesis, pulmonary arteries from broiler and Leghorn chickens were isolated and exposed to normoxia or hypoxia in the presence or absence of L-arginine while their constriction and relaxation responses to vasoactive compounds were recorded. In broilers, hypoxia did not affect the constriction responses of isolated pulmonary arteries but decreased EDNO-dependent acetylcholine-induced relaxation responses. In contrast, in Leghorns hypoxia increased endothelin-1-induced vasoconstriction responses and reduced the EDNO-dependent relaxation responses only to the lowest concentration of acetylcholine used. L-Arginine supplementation augmented the relaxation responses to acetylcholine in broilers and Leghorns under normoxia but failed to augment them under hypoxia. Relaxation responses to the NO donor, sodium nitroprusside, were not affected by hypoxia in Leghorns but were increased by hypoxia in broilers. These results suggest that the increased incidence of PHS in broiler chickens reared under hypoxia may be associated with a hypoxia-induced reduction in the synthesis or activity of EDNO in the pulmonary circulation.

Asunto(s)

Pollos , Contracción Muscular , Relajación Muscular , Músculo Liso Vascular/fisiología , Oxígeno/administración & dosificación , Arteria Pulmonar/fisiología , Acetilcolina/farmacología , Animales , Arginina/administración & dosificación , Peso Corporal , Endotelina-1/farmacología , Endotelio Vascular/metabolismo , Ventrículos Cardíacos/anatomía & histología , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/veterinaria , Hipoxia , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Óxido Nítrico/fisiología , Donantes de Óxido Nítrico/administración & dosificación , Nitroprusiato/administración & dosificación , Tamaño de los Órganos , Cloruro de Potasio/farmacología , Enfermedades de las Aves de Corral/etiología , Arteria Pulmonar/efectos de los fármacos

RESUMEN

This commentary presents the proceedings of the symposium sponsored by Cardiovascular Section of American Physiological Society in San Diego, CA on 12 April 2003. The major focus of this symposium was on the actions and physiological relevance of several novel Ca2+ signalling mechanisms in vascular smooth muscle (VSM) cells. Five important topics were presented in this symposium including the discovery and roles of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) in mediating Ca2+ release, Ca2+ sparks and activation of plasma membrane KCa channels in VSM cells, the role of cADPR-mediated activation of ryanodine receptors in the control of vascular tone, the role of [Ca2+]i in mechanotransduction in the arterioles, and interactions of mitochondrial Ca2+ release and SR Ca2+ mobilization. The purpose of this symposium was to promote discussions and exchange of ideas between scientists with interests in Ca2+ signalling mechanisms and those with interests in vascular physiology and pharmacology. The cross-fertilization of ideas is expected to greatly advance our understanding of the physiological and pharmacological relevance of these new Ca2+ signalling mechanisms.

Asunto(s)

Señalización del Calcio/fisiología , Músculo Liso Vascular/metabolismo , Arteriolas/metabolismo , Transporte Biológico , Calcio/metabolismo , ADP-Ribosa Cíclica/metabolismo , Humanos , Integrinas/metabolismo , Mitocondrias/metabolismo , NADP/análogos & derivados , NADP/metabolismo , Canales de Potasio/metabolismo

RESUMEN

Integrin binding by Arg-Gly-Asp (RGD)-containing peptides has been shown to alter vascular tone in a variety of blood vessels and has been implicated as a mechanism of vasoregulation during tissue injury. However, the effect of these peptides in the coronary circulation has not been examined. Thus the purpose of our study was to test the hypothesis that integrins act as receptors linked to the regulation of coronary vasomotor function. In particular, the ability of RGD-containing peptides to influence vascular tone by interacting with the alpha(v)beta(3)- and alpha(5)beta(1)-integrins was studied in isolated pig coronary arterioles. All vessels developed basal tone and dilated in a concentration-dependent manner to soluble peptides cyclic GPenGRGDSPCA (cyclic RGD), an alpha(v)beta(3)-cyclic-binding peptide (XJ735), DMP7677, an alpha(5)beta(1)-binding peptide, and to protease-generated (neutrophil elastase) fragments of denatured collagen type I (a major RGD-containing extracellular matrix protein). The vasodilations to cyclic RGD, XJ735, and collagen fragments were almost completely blocked by endothelial removal or by the cyclooxygenase inhibitor indomethacin. In contrast, after endothelial removal and incubation with indomethacin, coronary arterioles showed concentration-dependent constriction to the alpha(5)beta(1)-integrin ligand DMP7677 but not to cyclic RGD or XJ735. Collectively, our results indicate that activation of endothelial alpha(v)beta(3)- and alpha(5)beta(1)-integrins mediates coronary arteriolar dilation via the endothelial production of cyclooxygenase-derived prostaglandins. These data support a role for integrins in the regulation of coronary vascular tone that may be particularly important during myocardial injury.

Asunto(s)

Circulación Coronaria/fisiología , Oligopéptidos/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Receptores de Vitronectina/metabolismo , Vasodilatación/fisiología , Animales , Proteínas Portadoras/farmacología , Colágeno/farmacología , Colágeno Tipo I/farmacología , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Integrinas/metabolismo , Masculino , Microcirculación/efectos de los fármacos , Microcirculación/fisiología , Músculo Liso Vascular/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Péptidos Cíclicos/farmacología , Porcinos , Resistencia Vascular/efectos de los fármacos , Resistencia Vascular/fisiología , Vasodilatación/efectos de los fármacos

RESUMEN

The smooth muscle of arterioles responds to an increase in intraluminal pressure with vasoconstriction and with vasodilation when pressure is decreased. Such myogenic vasoconstriction provides a level of basal tone that enables arterioles to appropriately adjust diameter in response to neurohumoral stimuli. Key in this process of mechanotransduction is the role of changes in intracellular Ca(2+). However, it is becoming clear that considerable complexity exists in the spatiotemporal characteristics of the Ca(2+) signal and that changes in intracellular Ca(2+) may play roles other than direct effects on the contractile process via activation of myosin light-chain phosphorylation. The involvement of Ca(2+) may extend to modulation of ion channels and release of Ca(2+) from the sarcoplasmic reticulum, alterations in Ca(2+) sensitivity, and coupling between cells within the vessel wall. The purpose of this brief review is to summarize the current literature relating to Ca(2+) and the arteriolar myogenic response. Consideration is given to coupling of Ca(2+) changes to the mechanical stimuli, sources of Ca(2+), involvement of ion channels, and spatiotemporal aspects of intracellular Ca(2+) signaling.

Asunto(s)

Arteriolas/fisiología , Señalización del Calcio/fisiología , Músculo Liso Vascular/fisiología , Animales , Humanos , Canales Iónicos/fisiología , Retículo Sarcoplasmático/fisiología , Transducción de Señal/fisiología , Vasoconstricción/fisiología , Vasodilatación/fisiología

RESUMEN

The L-type calcium channel is the major calcium influx pathway in vascular smooth muscle and is regulated by integrin ligands, suggesting an important link between extracellular matrix and vascular tone regulation in tissue injury and remodeling. We examined the role of integrin-linked tyrosine kinases and focal adhesion proteins in regulation of L-type calcium current in single vascular myocytes. Soluble tyrosine kinase inhibitors blocked the increase in current produced by alpha(5) integrin antibody or fibronectin, whereas tyrosine phosphatase inhibition enhanced the effect. Cell dialysis with an antibody to focal adhesion kinase or with FRNK, the C-terminal noncatalytic domain of focal adhesion kinase, produced moderate (24 or 18%, respectively) inhibition of basal current but much greater inhibition (63 or 68%, respectively) of integrin-enhanced current. A c-Src antibody and peptide inhibitors of the Src homology-2 domain or a putative Src tyrosine phosphorylation site on the channel produced similar inhibition. Antibodies to the cytoskeletal proteins paxillin and vinculin, but not alpha-actinin, inhibited integrin-dependent current by 65-80%. Therefore, alpha(5)beta(1) integrin appears to regulate a tyrosine phosphorylation cascade involving Src and various focal adhesion proteins that control the function of the L-type calcium channel. This interaction may represent a novel mechanism for control of calcium influx in vascular smooth muscle and other cell types.

Asunto(s)

Canales de Calcio Tipo L/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Fibronectina/metabolismo , Transducción de Señal , Actinina/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Adhesión Celular , Proteínas del Citoesqueleto/farmacología , Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Matriz Extracelular/metabolismo , Fibronectinas/farmacología , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Integrinas/metabolismo , Ligandos , Masculino , Microscopía Fluorescente , Miocardio/citología , Técnicas de Placa-Clamp , Paxillin , Fosfoproteínas/farmacología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/biosíntesis , Ratas , Ratas Sprague-Dawley , Estilbenos/farmacología , Factores de Tiempo , Tirosina/metabolismo , Vinculina/farmacología

RESUMEN

This review summarizes what is currently known about the role of integrins in the vascular myogenic response. The myogenic response is the rapid and maintained constriction of a blood vessel in response to pressure elevation. A role for integrins in this process has been suggested because these molecules form an important mechanical link between the extracellular matrix and the vascular smooth muscle cytoskeleton. We briefly summarize evidence for a general role of integrins in mechanotransduction. We then describe the integrin subunit combinations known to exist in smooth muscle and the vascular wall matrix proteins that may interact with these integrins. We then discuss the effects of integrin-specific peptides and antibodies on vascular tone and on calcium entry mechanisms in vascular smooth muscle. Because integrin function is linked to the cytoskeleton, we discuss evidence for the role of the cytoskeleton in determining myogenic responsiveness. Finally, we analyze evidence that integrin-linked signaling pathways, such as those involving protein tyrosine phosphorylation cascades and mitogen-activated protein kinases, are required for myogenic tone.

Asunto(s)

Integrinas/fisiología , Mecanorreceptores/fisiología , Músculo Liso Vascular/fisiología , Transducción de Señal/fisiología , Animales , Presión Sanguínea , Calcio/fisiología , Citoesqueleto/fisiología , Matriz Extracelular/fisiología , Humanos

RESUMEN

Extracellular matrix (ECM) is known to provide signals controlling cell shape, migration, proliferation, differentiation, morphogenesis, and survival. Recent data shows that some of these signals are derived from biologically active cryptic sites within matrix molecules (matricryptic sites) that are revealed after structural or conformational alteration of these molecules. We propose the name, matricryptins, for enzymatic fragments of ECM containing exposed matricryptic sites. Mechanisms regulating the exposure of matricryptic sites within ECM molecules include the major mechanism of enzymatic breakdown as well as others including ECM protein multimerization, adsorption to other molecules, cell-mediated mechanical forces, and ECM denaturation. Such matrix alterations occur during or as a result of tissue injury, and thus, the appearance of matricryptic sites within an injury site may provide important new signals to regulate the repair process. Here, we review the data supporting this concept and provide insight into why the increased exposure of matricryptic sites may be an important regulatory step in tissue responses to injury.

Asunto(s)

Sitios de Unión/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Humanos , Oligopéptidos/metabolismo , Heridas y Lesiones/metabolismo , Heridas y Lesiones/fisiopatología

RESUMEN

Microtubules are important cytoskeletal elements that have been shown to play a major role in many cellular processes because of their mechanical properties and/or their participation in various cell signaling pathways. We tested the hypothesis that depolymerization of microtubules would alter vascular smooth muscle (VSM) tone and hence contractile function. In our studies, isolated cremaster arterioles exhibited significant vasoconstriction that developed over a 20- to 40-min period when they were treated with microtubule depolymerizing drugs colchicine (10 microM), nocodazole (10 microM), or demecolcine (10 microM). Immunofluorescent labeling of microtubules in cultured rat VSM revealed that both colchicine and nocodazole caused microtubule depolymerization over a similar time course. The vasoconstriction was maintained over a wide range of intraluminal pressures (30-170 cmH(2)O). The increased tone was not affected by endothelial denudation, suggesting that it was due to an effect on VSM. Microtubule depolymerization with demecolcine or colchicine had no effect on VSM intracellular Ca(2+) concentration ([Ca(2+)](i)). These data indicate that microtubules significantly interact with processes leading to the expression of vasomotor tone. The mechanism responsible for the effect of microtubules on vasomotor tone appears to be independent of both the endothelium and an increase in VSM [Ca(2+)](i).

Asunto(s)

Arteriolas/fisiología , Microtúbulos/metabolismo , Tono Muscular/fisiología , Músculo Liso Vascular/fisiología , Sistema Vasomotor/fisiología , Animales , Calcio/metabolismo , Colchicina/farmacología , Demecolcina/farmacología , Relación Dosis-Respuesta a Droga , Masculino , Microtúbulos/efectos de los fármacos , Nocodazol/farmacología , Polímeros/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

To localize activated protein kinase C (PKC) in smooth muscle cells, an antibody directed to the catalytic site of the enzyme was used to assess PKC distribution by immunofluorescence techniques in gastric smooth muscle cells isolated from Bufo marinus. An antibody to vinculin was used to delineate the cell membrane. High-resolution three-dimensional images of immunofluorescence were obtained from a series of images collected through focus with a digital imaging microscope. Cells were untreated or treated with agents that increase PKC activity (10 microM carbachol for 1 min, 1 microM phorbol 12-myristate 13-acetate (PMA) for 10 min), or have no effect on PKC activity (1 micrometer 4-alpha phorbol, 12,13-didecanoate (4-alpha PMA)). In unstimulated cells, activated PKC and vinculin were located and organized at the cell surface. Cell cytosol labeling for activated PKC was sparse and diffuse and was absent for vinculin. After treatment with carbachol, which stimulates contraction and PKC activity, in addition to the membrane localization, the activated PKC exhibited a pronounced cytosolic fibrillar distribution and an increased total fluorescence intensity relative to vinculin. The distributions of activated PKC observed after PMA but not 4-alpha PMA were similar to those observed with carbachol. Our results indicate that in resting cells there is a pool of activated PKC near the cell membrane, and that after stimulation activated PKC is no longer membrane-confined, but is present throughout the cytosol. Active PKC appears to associate with contractile filaments, supporting a possible role in modulation of contraction.

Asunto(s)

Músculo Liso/enzimología , Proteína Quinasa C/metabolismo , Animales , Bufo marinus , Carbacol/farmacología , Citosol/enzimología , Activación Enzimática/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Microscopía Fluorescente , Contracción Muscular/fisiología , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Ésteres del Forbol/farmacología , Estómago/citología , Estómago/efectos de los fármacos , Estómago/enzimología , Acetato de Tetradecanoilforbol/farmacología , Vinculina/metabolismo

RESUMEN

OBJECTIVE: To determine if the enhanced pressure-induced constriction of arterioles isolated from hypertensive rats is mediated by the endothelium. METHODS: We utilized isolated, cannulated first-order arterioles (80 to 110 microns, i.d.) from the rat cremaster muscle of spontaneously hypertensive (SHR) and normotensive (WKY) rats. Arteriolar diameter was measured in response to changes in intraluminal pressures as well as various pharmacological agents in the presence and absence of an intact endothelial cell lining. RESULTS: All arterioles developed intrinsic tone (approximately 74% of passive). Pressure-diameter relationships over a pressure range of 30 to 170 cm H2O demonstrated that the myogenic response of arterioles derived from both WKY and SHR was not dependent upon an intact endothelium. However, at higher pressures (> 170 cm H2O) the ability of the denuded arteriole from the SHR to maintain a constricted diameter was completely abolished. Treatment of arterioles from the SHR having intact endothelium with diclofenac (10(-5) M; 30 min) to inhibit the effect of cyclooxygenase had no effect on the high pressure constriction. In contrast, application of BQ-123 (10(-7) M; 30 min), an ET-A receptor blocker used to inhibit vascular smooth muscle responses to endothelin, completely abolished the arteriolar constriction at higher pressures. CONCLUSIONS: Therefore, in the hypertensive, arteriolar vasomotor responses to changes in intraluminal pressure is due to at least two mechanisms; one that is intrinsic to vascular smooth muscle (i.e., myogenic) and a second that involves an endothelial cell release of endothelin.

Asunto(s)

Arteriolas/metabolismo , Endotelinas/metabolismo , Hipertensión/fisiopatología , Animales , Antiinflamatorios no Esteroideos/farmacología , Arteriolas/efectos de los fármacos , Técnicas de Cultivo , Diclofenaco/farmacología , Masculino , Microscopía por Video , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY

RESUMEN

Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by alphav beta3 and alpha5 beta1 integrins. To examine the underlying mechanisms, we measured L-type Ca2+ channel current in arteriolar smooth muscle cells in response to integrin ligands. Whole-cell, inward Ba2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti-beta3 integrin antibodies, or monovalent beta3 antibody. With VN or beta3 antibody coated onto microbeads and presented as an insoluble ligand, current was also inhibited. In contrast, beads coated with FN or alpha5 antibody produced significant enhancement of current after bead attachment. Soluble alpha5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in combination with an appropriate IgG. The data suggest that alphavbeta3 and alpha5 beta1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury.

Asunto(s)

Arteriolas/fisiología , Canales de Calcio/fisiología , Músculo Liso Vascular/fisiología , Receptores de Fibronectina/fisiología , Receptores de Vitronectina/fisiología , Animales , Anticuerpos/farmacología , Anticuerpos Monoclonales/farmacología , Arteriolas/citología , Canales de Calcio/efectos de los fármacos , Canales de Calcio Tipo L , Fibronectinas/farmacología , Inmunoglobulina G/farmacología , Técnicas In Vitro , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Músculo Liso Vascular/citología , Oligopéptidos/farmacología , Ratas , Ratas Sprague-Dawley , Receptor Cross-Talk/efectos de los fármacos , Receptor Cross-Talk/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Vitronectina/farmacología

RESUMEN

Integrins are transmembrane adhesion receptors found on most cells, including vascular smooth muscle cells. Several integrins bind to the conserved amino acid sequence Arg-Gly-Asp (RGD), and synthetic RGD-containing peptides can cause endothelium-independent arteriolar vasodilation by interacting with the alphavbeta3-integrin expressed by vascular smooth muscle. We hypothesized that RGD peptide-induced vasodilation involves K+ channels. Rat cremaster arterioles were treated with cRGD (GPenGRGDSPCA) in the presence or absence of the nonselective K+ channel inhibitor tetraethylammonium (TEA, 20 mM). TEA caused arterioles to constrict by 19 +/- 5% and inhibited cRGD-induced vasodilation (n = 7, P < 0.05). Vessels preconstricted with phenylephrine (5 x 10(-7) M) showed no significant inhibition of the dilatory response to cRGD, indicating that inhibition by TEA was not related to increased vasomotor tone. Further evidence for the involvement of K+ channels was obtained by addition of 100 mM KCl (n = 5), which inhibited vasodilation caused by cRGD. Inhibition of large and small conductance, Ca2+-activated K+ channels with iberiotoxin (100 nM) or apamin (25 nM), respectively, had no effect on cRGD-induced vasodilation. Partial inhibition of vasodilation was observed with inhibitors of voltage-gated (4-aminopyridine, 1 mM), ATP-sensitive (glibenclamide, 1 microM), and inward rectifying (barium, 50 microM) K+ channels. These data support the hypothesis that integrin-signaling pathways leading to arteriolar vasodilation may involve modulation of K+ channel function.

Asunto(s)

Arteriolas/fisiología , Integrinas/fisiología , Oligopéptidos/farmacología , Canales de Potasio/fisiología , Vasodilatación/fisiología , Animales , Arteriolas/efectos de los fármacos , Técnicas In Vitro , Integrinas/efectos de los fármacos , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Bloqueadores de los Canales de Potasio , Canales de Potasio/efectos de los fármacos , Cloruro de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Tetraetilamonio/farmacología , Vasodilatación/efectos de los fármacos

RESUMEN

Recent work has shown that osteopontin expression is upregulated at sites of cardiovascular injury. It has been hypothesized that osteopontin provides an adhesive matrix for endothelial and smooth muscle cells during remodeling of the vascular wall following injury. Osteopontin has also been found to be synthesized by monocytes and macrophages within injury sites. Here, we present data showing that osteopontin can promote leukocyte adhesion through the alpha4beta1 integrin. In the presence of physiologic concentrations of Mg2+ and Ca2+, osteopontin purified from bovine milk promoted cell-substrate adhesion of HL-60 and Ramos cells, two model leukocyte cell lines. As with other adhesive ligands, adhesion to osteopontin required leukocyte activation. Under these conditions, no adhesion to control substrates such as bovine serum albumin was observed. Leukocyte adhesion was inhibited by anti-integrin antibodies directed at either the alpha4 or beta1 integrin subunits but not by control antibodies directed to other integrins. Further adhesion experiments revealed that leukocyte binding to osteopontin was completely inhibited by an alpha4beta1-binding peptide containing the leucine-aspartate-valine (LDV) sequence, while a control, non-binding peptide containing leucine-glutamate-valine (LEV) had minimal effects. Affinity chromatography using either surface labeled HL-60 or Ramos cell extracts revealed that the alpha4beta1 integrin specifically bound to osteopontin. Immunoprecipitation of eluted fractions from these columns positively identified the alpha4beta1 integrin. In order to localize potential alpha4beta1-binding sites within osteopontin, the protein was proteolytically cleaved with thrombin. A 30 kDa N-terminal osteopontin fragment purified using fast protein liquid chromatography promoted alpha4beta1 dependent leukocyte adhesion in a manner similar to that of the intact protein. These data collectively demonstrate that the alpha4beta1 integrin is a new adhesion receptor for osteopontin and that an alpha4beta1 binding site exists in the NH2-terminal thrombin fragment of osteopontin.

Asunto(s)

Integrinas/metabolismo , Leucocitos/efectos de los fármacos , Receptores Mensajeros de Linfocitos/metabolismo , Sialoglicoproteínas/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Vasos Sanguíneos/lesiones , Bovinos , Adhesión Celular/efectos de los fármacos , Línea Celular , Cromatografía de Afinidad , Células HL-60/efectos de los fármacos , Lesiones Cardíacas/fisiopatología , Humanos , Integrina alfa4beta1 , Integrinas/antagonistas & inhibidores , Integrinas/inmunología , Leucocitos/metabolismo , Ligandos , Osteopontina , Unión Proteica , Receptores Mensajeros de Linfocitos/antagonistas & inhibidores , Receptores Mensajeros de Linfocitos/inmunología , Albúmina Sérica Bovina/metabolismo , Sialoglicoproteínas/farmacología , Trombina/farmacología , Cicatrización de Heridas/fisiología

RESUMEN

The ability of an integrin-binding Arg-Gly-Asp-Asn (RGDN)- containing peptide to influence vascular tone by interacting with the alpha5beta1 integrin was studied using rat skeletal muscle arterioles. After blockade of beta3 integrin function, isolated arterioles with spontaneous tone showed concentration-dependent vasoconstrictions to topical application of GRGDNP, a peptide that shows a greater ability to interact with alpha5beta1 than with alphavbeta3. The constriction to GRGDNP (2.1 mM) was inhibited by blocking alpha5 integrin function, and was intensified by blocking beta3 integrin function. In contrast, GRGDSP, a peptide that interacts better with alphavbeta3, was unable to induce sustained constrictions. Removal of the endothelium abolished the vasoconstriction in response to GRGDNP, suggesting that the response was due to release of an endothelium-dependent factor. Indeed, blockade of ETA endothelin receptors with BQ-610 (1 microM), similar to removal of the endothelium and alpha5 integrin blockade, inhibited the vasoconstriction. These data indicate that interaction of RGD peptides, and in particular the RGDN sequence with endothelial cell alpha5beta1, causes endothelin-mediated arteriolar vasoconstriction. These results indicate that integrins are novel signaling receptors within the vascular wall that affect vasomotor tone, and may play an important role in vascular control.

Asunto(s)

Arteriolas/fisiología , Antagonistas de los Receptores de Endotelina , Endotelio Vascular/fisiología , Músculo Esquelético/irrigación sanguínea , Oligopéptidos/farmacología , Receptores de Fibronectina/fisiología , Animales , Anticuerpos Bloqueadores/farmacología , Antígenos CD/inmunología , Arteriolas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Fibronectinas/farmacología , Técnicas In Vitro , Integrina alfa5 , Integrina beta3 , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Inhibidores de Agregación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/inmunología , Ratas , Ratas Sprague-Dawley , Receptor de Endotelina A , Receptores de Vitronectina/fisiología , Factores de Tiempo , Vasoconstricción/fisiología

RESUMEN

The purpose of this study was to measure vascular smooth muscle (VSM) cytosolic calcium ([Ca2+]i) during the myogenic response. We examined the temporal and steady-state relationships between lumen diameter and VSM [Ca2+]i in isolated arterioles exposed to step changes in intravascular pressure. We also studied the relationship between step sizes in intravascular pressure and changes in [Ca2+]i. First-order arterioles from the hamster cheek pouch were isolated, cannulated, and pressurized. [Ca2+]i was quantified using the ratio of emitted fluorescence intensity (R340/380) during alternate excitation of fura 2-loaded vessels at 340 and 380 nm. Stepwise increases in transmural pressure elicited corresponding increases in steady-state [Ca2+]i and myogenic constriction. From a common baseline pressure, the initial rise in [Ca2+]i after a step change in pressure was directly related to the magnitude of the step size and of the distension caused by that pressure step. This supports the theory that there is a relationship between the initial distension of the vessel and the initial [Ca2+]i change. Also, increasing the size of the step change in pressure resulted in a greater myogenic response, yet no difference in the steady-state [Ca2+]i was detected, which suggests that Ca2+ is not the principal or only determinant of steady-state constriction. Finally, larger increases in [Ca2+]i do not necessarily augment the myogenic response, which suggests that some minimal rise in [Ca2+]i is required to elicit myogenic vasoconstriction. Collectively, these data suggest the presence of a separate, Ca(2+)-independent regulatory system.

Asunto(s)

Arteriolas/fisiología , Calcio/metabolismo , Músculo Liso Vascular/fisiología , Vasoconstricción/fisiología , Animales , Arteriolas/efectos de los fármacos , Presión Sanguínea , Cricetinae , Técnicas In Vitro , Masculino , Mesocricetus , Tono Muscular , Músculo Liso Vascular/efectos de los fármacos , Fenilefrina/farmacología , Transducción de Señal , Factores de Tiempo , Vasoconstricción/efectos de los fármacos