RESUMEN
Vascular endothelial cells are known to respond to a range of biochemical and time-varying mechanical cues that can promote blood vessel sprouting termed angiogenesis. It is less understood how these cells respond to sustained (i.e., static) mechanical cues such as the deformation generated by other contractile vascular cells, cues which can change with age and disease state. Here we demonstrate that static tensile strain of 10%, consistent with that exerted by contractile microvascular pericytes, can directly and rapidly induce cell cycle re-entry in growth-arrested microvascular endothelial cell monolayers. S-phase entry in response to this strain correlates with absence of nuclear p27, a cyclin-dependent kinase inhibitor. Furthermore, this modest strain promotes sprouting of endothelial cells, suggesting a novel mechanical 'angiogenic switch'. These findings suggest that static tensile strain can directly stimulate pathological angiogenesis, implying that pericyte absence or death is not necessarily required of endothelial cell re-activation.
Asunto(s)
Ciclo Celular , Células Endoteliales/fisiología , Neovascularización Fisiológica , Pericitos/fisiología , Resistencia a la Tracción , Animales , Fenómenos Biomecánicos , Humanos , Estrés MecánicoRESUMEN
BACKGROUND: Propranolol, a ß-adrenergic receptor (AR) antagonist, is an effective treatment for endangering infantile haemangioma (IH). Dramatic fading of cutaneous colour is often seen a short time after initiating propranolol therapy, with accelerated regression of IH blood vessels discerned after weeks to months. OBJECTIVES: To assess a possible role for haemangioma-derived pericytes (HemPericytes) isolated from proliferating and involuting phase tumours in apparent propranolol-induced vasoconstriction. METHODS: HemPericytes were assayed for contractility on a deformable silicone substrate: propranolol (10 µmol L(-1)) restored basal contractile levels in HemPericytes that were relaxed with the AR agonist epinephrine. Small interfering RNA knockdown of ß2-AR blunted this response. HemPericytes and haemangioma-derived endothelial cells were co-implanted subcutaneously in nude mice to form blood vessels; at day 7 after injection, mice were randomized into vehicle and propranolol-treated groups. RESULTS: HemPericytes expressed high levels of ß2-AR mRNA compared with positive control bladder smooth muscle cells. In addition, ß2-AR mRNA levels were relatively high in IH specimens (n = 15) compared with ß1-AR, ß3-AR and α1b-AR. Normal human retinal and placental pericytes were not affected by epinephrine or propranolol in this assay. Propranolol (10 µmol L(-1)) inhibited the proliferation of HemPericytes in vitro, as well as normal pericytes, indicating a nonselective effect in this assay. Contrast-enhanced microultrasonography of the implants after 7 days of treatment showed significantly decreased vascular volume in propranolol-treated animals, but no reduction in vehicle-treated animals. CONCLUSIONS: These findings suggest that the mechanism of propranolol's effect on proliferating IH involves increased pericytic contractility.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Hemangioma/fisiopatología , Pericitos/fisiología , Propranolol/farmacología , Vasoconstricción/efectos de los fármacos , Animales , Volumen Sanguíneo , Proliferación Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Epinefrina/farmacología , Hemangioma/irrigación sanguínea , Humanos , Masculino , Ratones Desnudos , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Distribución Aleatoria , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Trasplante Heterólogo , Células Tumorales Cultivadas , Vasoconstrictores/farmacología , Vasodilatadores/farmacologíaRESUMEN
The actin supergene family encodes a number of structurally related, but perhaps functionally distinct, protein isoforms that regulate contractile potential in muscle tissues and help to control the shape as well as the motility of non-muscle cells. In spite of the documented conservation amongst isoactin genes and their encoded proteins, recent results of biochemical, antibody localization, molecular mutagenesis and isoactin gene replacement studies lend credence to the notion that functional differences amongst muscle and non-muscle actin isoforms exist. Furthermore, the discovery of a new class of actin isoforms, the actin-related proteins, reveals that the actin gene and protein isoform family is more complex than was previously believed.
Asunto(s)
Actinas/clasificación , Actinas/genética , Actinas/fisiología , Secuencia de Aminoácidos , Animales , Movimiento Celular , Secuencia de Consenso , Citoesqueleto/ultraestructura , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Estructura Molecular , Familia de Multigenes , Músculos/metabolismo , Especificidad de Órganos , Procesamiento Proteico-Postraduccional , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
We have affinity-fractionated rabbit antiactin immunoglobulins (IgG) into classes that bind preferentially to either muscle or nonmuscle actins. The pools of muscle- and nonmuscle-specific actin antibodies were used in conjunction with fluorescence microscopy to characterize the actin in vascular pericytes, endothelial cells (EC), and smooth muscle cells (SMC) in vitro and in situ. Nonmuscle-specific antiactin IgG stained the stress fibers of cultured EC and pericytes but did not stain the stress fibers of cultured SMC, although the cortical cytoplasm associated with the plasma membrane of SMC did react with nonmuscle-specific antiactin. Whereas the muscle-specific antiactin IgG failed to stain EC stress fibers and only faintly stained their cortical cytoplasm, these antibodies reacted strongly with the fiber bundles of cultured SMC and pericytes. Similar results were obtained in situ. The muscle-specific antiactin reacted strongly with the vascular SMC of arteries and arterioles as well as with the perivascular cells (pericytes) associated with capillaries and post-capillary venules. The non-muscle-specific antiactin stained the endothelium and the pericytes but did not react with SMC. These findings indicate that pericytes in culture and in situ possess both muscle and nonmuscle isoactins and support the hypothesis that the pericyte may represent the capillary and venular correlate of the SMC.
Asunto(s)
Actinas/metabolismo , Endotelio/metabolismo , Microcirculación/citología , Músculo Liso Vascular/metabolismo , Actinas/inmunología , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Arterias/citología , Arteriolas/citología , Capilares/citología , Bovinos , Citoesqueleto/metabolismo , Endotelio/ultraestructura , Técnica del Anticuerpo Fluorescente , Punto Isoeléctrico , Microcirculación/metabolismo , Músculo Liso Vascular/ultraestructura , Distribución Tisular , Vénulas/citologíaRESUMEN
We characterized the form and distribution of muscle and nonmuscle actin within retinal pericytes. Antibodies with demonstrable specificities for the actin isoforms were used in localization and immunoprecipitation experiments to identify those cellular domains that were enriched or deficient in one or several actin isoforms. Living pericyte behavior was monitored with phase-contract video microscopy before fixation to identify those cellular areas that might preferentially be stained with either of the fluorescent antiactins or phallotoxins. Antibody and phallotoxin staining of pericytes revealed that nonmuscle actin is present within membrane ruffles, pseudopods, and stress fibers. In contrast, muscle actin could be convincingly localized in stress fibers, but not within specific motile areas of pericyte cytoplasm. To confirm and quantitatively extend the results obtained by fluorescence microscopy, nonionic and ionic detergents were used to selectively extract the motile or immobilized (stress fiber-containing) regions of biosynthetically labeled pericyte cytoplasm. Immunoprecipitated actins that were present within these discrete cellular domains were subjected to isoelectric focusing in urea-polyacrylamide gels before fluorographic analysis. Scanning laser densitometry of the focused actins could not reveal any detectable alpha-actin within those beta- and gamma-actin-enriched motile regions extracted with nonionic detergents. Moreover, when pericyte stress fibers are completely dissolved by ionic detergent lysis, three actin isoforms can be quantified to be present in a ratio of 1:2.75:3 (alpha:beta:gamma). These biochemical findings on biosynthetically labeled and immunoprecipitated pericyte actins confirm the fluorescent localization studies. While the regulatory events governing this actin sorting are unknown, it seems possible that such events may play important roles in controlling cell shape, adhesion, or the promotion of localized cell spreading.
Asunto(s)
Actinas/metabolismo , Microcirculación/ultraestructura , Citoesqueleto de Actina/ultraestructura , Animales , Bovinos , Compartimento Celular , Movimiento Celular , Citoesqueleto/ultraestructura , Técnicas In Vitro , Punto Isoeléctrico , Microscopía Fluorescente , Pruebas de Precipitina , Retina/irrigación sanguínea , Retina/citología , Grabación en VideoRESUMEN
Whereas it has been demonstrated that muscle and nonmuscle isoactins are segregated into distinct cytoplasmic domains, the mechanism regulating subcellular sorting is unknown (Herman, 1993a). To reveal whether isoform-specific actin-binding proteins function to coordinate these events, cell extracts derived from motile (Em) versus stationary (Es) cytoplasm were selectively and sequentially fractionated over filamentous isoactin affinity columns prior to elution with a KCl step gradient. A polypeptide of interest, which binds specifically to beta-actin filament columns, but not to muscle actin columns has been conclusively identified as the ERM family member, ezrin. We studied ezrin-beta interactions in vitro by passing extracts (Em) over isoactin affinity matrices in the presence of Ca(2+)-containing versus Ca(2+)-free buffers, with or without cytochalasin D. Ezrin binds and can be released from beta-actin Sepharose-4B in the presence of Mg2+/EGTA and 100 mM NaCl (at 4 degrees C and room temperature), but not when affinity fractionation of Em is carried out in the presence of 0.2 mM CaCl2 or 2 microM cytochalasin D. N-acetyl-(leucyl)2-norleucinal and E64, two specific inhibitors of the calcium-activated protease, calpain I, protect ezrin binding to beta actin in the presence of calcium. Moreover, biochemical analysis of endothelial lysates reveals that a calpain I cleavage product of ezrin emerges when cell locomotion is stimulated in response to monolayer injury. Immunofluorescence analysis of leading lamellae reveals that anti-ezrin and anti-beta-actin IgGs can be simultaneously co-localized, extending the results of isoactin affinity fractionation of Em-derived extracts and suggesting that ezrin and beta-actin interact in vivo. To test the hypothesis that ezrin binds directly to beta-actin, we performed three sets of studies under a wide range of physiological conditions (pH 7.0-8.5) using purified pericyte ezrin and either alpha- or beta-actin. These included co-sedimentation, isoactin affinity fractionation, and co-immunoprecipitation. Results of these experiments reveal that purified ezrin does not directly bind to beta-actin filaments, either in solution or while isoactins are covalently cross-linked to Sepharose-4B. This is in contrast to our finding that ezrin and beta-actin could be co-immunoprecipitated or co-sedimented from Em-derived cell lysates. To explore whether calcium transients occur in cellular domains enriched in ezrin and beta-actin, we mapped cellular free calcium in endothelial monolayers crawling in response to injury.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Actinas/metabolismo , Calcio/metabolismo , Movimiento Celular/fisiología , Citosol/metabolismo , Fosfoproteínas/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Vasos Sanguíneos/citología , Bovinos , Reactivos de Enlaces Cruzados , Citocalasina D/farmacología , Proteínas del Citoesqueleto , Endotelio/citología , Endotelio/metabolismo , Unión Proteica/efectos de los fármacos , Vasos Retinianos/citologíaRESUMEN
We localized myosin in vertebrate nonmuscle cells by electron microscopy using purified antibodies coupled with ferritin. Native and formaldehyde-fixed filaments of purified platelet myosin filaments each consisting of approximately 30 myosin molecules bound an equivalent number of ferritin-antimyosin conjugates. In preparations of crude platelet actomyosin, the ferritin-antimyosin bound exclusively to similar short, 10-15 nm wide filaments. In both cases, binding of the ferritin-antimyosin to the myosin filaments was blocked by preincubation with unlabeled antimyosin. With indirect fluorescent antibody staining at the light microscope level, we found that the ferritin-antimyosin and unlabeled antimyosin stained HeLa cells identically, with the antibodies concentrated in 0.5-microns spots along stress fibers. By electron microscopy, we found that the concentration of ferritin-antimyosin in the dense regions of stress fibers was five to six times that in the intervening less dense regions, 20 times that in the cytoplasmic matrix, and 100 times that in the nucleus. These concentration differences may account for the light microscope antibody staining pattern of spread interphase cells. Some, but certainly not all, of the ferritin-antimyosin was associated with 10-15-nm filaments. In mouse intestinal epithelial cells, ferritin-antimyosin was located almost exclusively in the terminal web. In isolated brush borders exposed to 5 mM MgCl2, ferritin-antimyosin was also concentrated in the terminal web associated with 10-15-nm filaments.
Asunto(s)
Membrana Celular/análisis , Citoplasma/análisis , Citoesqueleto/análisis , Microvellosidades/análisis , Miosinas/análisis , Actomiosina/análisis , Anticuerpos , Plaquetas , Ferritinas , Células HeLa , Humanos , Microscopía Electrónica , Miosinas/inmunologíaRESUMEN
We purified actin antibodies by affinity chromatography from the serum of rabbits immunized with glutaraldehyde-fixed chicken gizzard actin filaments and used this anti-actin to localize actin in myofibrils and fixed cultured cells at each stage of the cell cycle. By double immunodiffusion the anti-actin reacted with both smooth and skeletal muscle actin. Several blocking and absorption experiments demonstrated that the antibodies also bound specifically to actin in nonmuscle cells. The same structures stained using either the direct or the indirect fluorescent antibody technique; and, while the indirect method was more sensitive, the direct method was superior because there was no detectable nonspecific staining. As expected, anti-actin stained the I-band of myofibrils. It also stained stress fibers and membrane ruffles in HeLa cells. Some PtK-2 cells have straight stress fibers which stained with anti-actin, but in confluent cultures all PtK-2 cells have, instead, sinuous phase-dense fibers which stained with antibody. At prophase the whole cytoplasm stained uniformly with anti-actin. During metaphase and anaphase, anti-actin staining was concentrated diffusely in the mitotic spindle. In contrast, fluorescent heavy meromyosin stained discrete fine spindle fibers in these fixed cells. During cytokinesis, anti-actin stained the whole cytoplasm uniformly and was not concentrated in the cleavage furrow.
Asunto(s)
Actinas/aislamiento & purificación , Ciclo Celular , Subfragmentos de Miosina/aislamiento & purificación , Actinas/inmunología , Animales , Anticuerpos/inmunología , Línea Celular , Técnica del Anticuerpo Fluorescente , Células HeLa , Inmunodifusión , Macropodidae , Miofibrillas/análisisRESUMEN
Previous work in our laboratory has shown that microvascular pericytes sort muscle and nonmuscle actin isoforms into discrete cytoplasmic domains (Herman, I. M., and P. A. D'Amore. 1985. J. Cell Biol. 101:43-52; DeNofrio, D.T.C. Hoock, and I. M. Herman. J. Cell. Biol. 109:191-202). Specifically, muscle (alpha-smooth) actin is present on the stress fibers while nonmuscle actins (beta and gamma) are located on stress fibers and in regions of moving cytoplasm (e.g., ruffles, lamellae). To determine the form and function of beta actin in microvascular pericytes and endothelial cells recovering from injury, we prepared isoform-specific antibodies and cDNA probes for immunolocalization, Western and Northern blotting, as well as in situ hybridization. Anti-beta actin IgG was prepared by adsorption and release of beta actin-specific IgG from electrophoretically purified pericyte beta actin bound to nitrocellulose paper. Anti-beta actin IgGs prepared by this affinity selection procedure showed exclusive binding to beta actin present in crude cell lysates containing all three actin isoforms. For controls, we localized beta actin as a bright rim of staining beneath the erythrocyte plasma membrane. Anti-beta actin IgG, absorbed with beta actin bound to nitrocellulose, failed to stain erythrocytes. Simultaneous localization of beta actin with the entire F-actin pool was performed on microvascular pericytes or endothelial cells and 3T3 fibroblasts recovering from injury using anti-beta actin IgG in combination with fluorescent phalloidin. Results of these experiments revealed that pericyte beta actin is localized beneath the plasma membrane in association with filopods, pseudopods, and fan lamellae. Additionally, we observed bright focal fluorescence within fan lamellae and in association with the ends of stress fibers that are preferentially associated with the ventral plasmalemma. Whereas fluorescent phalloidin staining along the stress fibers is continuous, anti-beta actin IgG localization is discontinuous. When injured endothelial and 3T3 cells were stained through wound closure, we localized beta actin only in motile cytoplasm at the wound edge. Staining disappeared as cells became quiescent upon monolayer restoration. Appearance of beta actin at the wound edge correlated with a two- to threefold increase in steady-state levels of beta actin mRNA, which rose within 15-60 min after injury and returned to noninjury levels during monolayer restoration. In situ hybridization revealed that transcripts encoding beta actin were localized at the wound edge in association with the repositioned protein. Results of these experiments indicate that beta actin and its encoded mRNA are polarized at the membrane-cytoskeletal interface within regions of moving cytoplasm.
Asunto(s)
Actinas/análisis , Membrana Celular/química , Citoplasma/química , Endotelio Vascular/química , Músculo Liso Vascular/química , Actinas/genética , Animales , Bovinos , Compartimento Celular , Movimiento Celular/fisiología , Células Cultivadas , Endotelio Vascular/lesiones , Eritrocitos/química , Fibroblastos/química , Humanos , Inmunoglobulina G/aislamiento & purificación , Inmunohistoquímica , Hibridación de Ácido Nucleico , ARN Mensajero/análisis , Cicatrización de Heridas/fisiologíaRESUMEN
We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.
Asunto(s)
Actinas/análisis , Movimiento Celular , Citoplasma/análisis , Miosinas/análisis , Animales , Membrana Celular/ultraestructura , Células Cultivadas , Embrión de Pollo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Interfase , Seudópodos/ultraestructura , Grabación de Cinta de VideoRESUMEN
Although there is considerable evidence to suggest that hemodynamics play an important role in vascular disease processes, the exact mechanisms are unknown. With this in mind, we have designed a pulsatile perfusion apparatus which reproducibly delivers pulsatile hemodynamics upon freshly excised canine carotid arteries in vitro. Quantifiable simulations included normotension with normal or lowered flow rates (120/80 mmHg, 120 and 40 ml/min), normotension with lowered or elevated transmural pressures (40-170 mmHg), and elevated pulse pressure (120 and 80 mmHg) with normal (150 ml/min) or elevated rates of flow (300 and 270 ml/min). Arterial biomechanical stresses and cellular behaviors were characterized biochemically and morphologically under all these stimulations which continued for 2-24 h. We found that increased pulse pressure alone had little effect on the total amount of radiolabeled [4-14C]cholesterol present within the medial compartment. However, normotension when coupled with altered transmural pressure yielded a three- to fourfold increase. Combinations of increased pulse pressure and flow potentiated cholesterol uptake by a factor of 10 when compared with normotension control values. Simulations that enhanced carotid arterial cholesterol uptake also influenced the endothelial cytoskeletal array of actin. Stress fibers were not present within the carotid endothelial cells of either the sham controls or the normotension and increased pulse pressure (normal flow) simulations. Endothelial cells lining carotids exposed to elevations in flow or those present within vessels perfused as per simulation b above assembled stress fibers (x = 4 and 10 per cell, respectively) within the time course of these studies. When endothelial cells were subjected to hemodynamic conditions that potentiated maximally cholesterol transport, no diffuse or stress fiber staining could be seen, but the cortical array of actin was intact. These results suggest that those biomechanical stresses that alter endothelial permeability and intimal integrity may do so via cytoskeletal actin signaling.
Asunto(s)
Velocidad del Flujo Sanguíneo , Presión Sanguínea , Arterias Carótidas/ultraestructura , Citoesqueleto/ultraestructura , Animales , Arterias Carótidas/metabolismo , Colesterol/metabolismo , Perros , Endotelio/ultraestructura , Hipertensión/metabolismo , Hipertensión/patología , Perfusión/instrumentación , Estrés MecánicoRESUMEN
Previous studies suggest that the Ca2+-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3- derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.
Asunto(s)
Actinas/metabolismo , Calpaína/metabolismo , Células 3T3 , Animales , Proteínas de Unión al Calcio/genética , Calpaína/antagonistas & inhibidores , Movimiento Celular , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas del Citoesqueleto , Regulación hacia Abajo , Humanos , Ratones , Fosfoproteínas/metabolismo , Especificidad por Sustrato , Regulación hacia ArribaRESUMEN
Fluorescence microscopy with 7-nitrobenz-2-oxa-3-diazole phallacidin was used to survey vertebrate tissues for actin filament bundles comparable to the stress fibers of cultured cells. Such bundles were found only in vascular endothelial cells. Like the stress fibers of cultured cells, these actin filament bundles were stained in a punctate pattern by fluorescent antibodies to both alpha-actinin and myosin. The stress fibers were oriented parallel to the direction of blood flow and were prominent in endothelial cells from regions exposed to high-velocity flow, such as the left ventricle, aortic valve, and aorta. Actin bundles may help the endothelial cell to withstand hemodynamic stress.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/ultraestructura , Endotelio/ultraestructura , Animales , Miosinas/metabolismoRESUMEN
Artery wall calcification associated with atherosclerosis frequently contains fully formed bone tissue including marrow. The cellular origin is not known. In this study, bone morphogenetic protein-2a, a potent factor for osteoblastic differentiation, was found to be expressed in calcified human atherosclerotic plaque. In addition, cells cultured from the aortic wall formed calcified nodules similar to those found in bone cell cultures and expressed bone morphogenetic protein-2a with prolonged culture. The predominant cells in these nodules had immunocytochemical features characteristic of microvascular pericytes that are capable of osteoblastic differentiation. Pericyte-like cells were also found by immunohistochemistry in the intima of bovine and human aorta. These findings suggest that arterial calcification is a regulated process similar to bone formation, possibly mediated by pericyte-like cells.
Asunto(s)
Arteriosclerosis/metabolismo , Proteínas/fisiología , Animales , Aorta , Proteínas Morfogenéticas Óseas , Bovinos , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Hibridación in Situ , Microscopía Electrónica , Músculo Liso Vascular/citología , Sondas ARNRESUMEN
Recent evidence indicates that basic fibroblast growth factor (bFGF), which lacks a conventional signal recognition sequence, is a component of the subendothelial matrix. However, the molecular mechanisms regulating its cellular release and subsequent matrix deposition remain equivocal. To examine the cellular and subcellular mechanisms regulating bFGF release and subendothelial sequestration, we generated polyclonal antibodies against a chemically cross-linked bFGF. We then used anti-bFGF IgG in conjunction with 3T3 cell [3H]thymidine incorporation assays, enzyme immunoassays and immunofluorescence to learn whether bFGF accumulation in the subendothelial matrix is dependent upon endothelial cell (EC)-cell contact, which coincides with growth arrest. In contrast to subconfluent cultures, which lacked any detectable extracellular matrix bFGF localization, bovine aortic and microvascular EC plated at confluent densities displayed a punctate extracellular staining pattern that was abolished when EC were pretreated with 10 micrograms/ml cycloheximide. Additionally, when EC were treated with either 1 mM beta-D xyloside, an inhibitor of proteoglycan assembly, or 100 micrograms/ml heparin, there was a 40% reduction in matrix-associated bFGF (quantified by image analysis of antibody stained cultures). 3T3 [3H]thymidine incorporation assays indicated that the beta-D xyloside-induced reduction of matrix-associated bFGF coincided with a significant increase in bFGF activity in the conditioned media. Neither sparsely-plated nor confluent EC cultures possessed specific bFGF localization of the nuclear compartment when cells were fixed using cold methanol; however, when EC were fixed in formaldehyde and lysed in isotonic buffers containing 0.1% Triton X-100 or absolute acetone, there was a marked decrease in anti-bFGF staining of the postconfluent extracellular matrix and a concomitant increase in nuclear fluorescence. Because bFGF-stimulated vascular cell growth has been implicated in controlling neointimal cell proliferation, we screened normal and atherosclerotic coronary blood vessels for bFGF, but we were unable to detect it either in lesioned or normal intima. In contrast, significant bFGF levels were observed in association with the EC and mesangial cells of the renal corpuscle, where heparan sulfate accumulates within the glomerular basement membrane. Our in vitro results suggest that bFGF accumulates within the proteoglycan-containing subendothelial matrix concomitant with the formation of cell-cell contacts. In situ, the composition of the microvascular matrix and the cellular phenotype may facilitate the selective accumulation of bFGF that we observed. This, in turn, may influence vascular morphogenesis and remodeling during angiogenesis.
Asunto(s)
Endotelio Vascular/metabolismo , Matriz Extracelular/química , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Células 3T3/metabolismo , Animales , Bovinos , Recuento de Células , Células Cultivadas/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/análisis , Glicósidos/farmacología , Heparina/farmacología , Glomérulos Renales/química , RatonesRESUMEN
Proton-translocating ATPases of the vacuolar class (V-ATPases) are found in a variety of animal cell compartments that participate in vesicular membrane transport, including clathrin-coated vesicles, endosomes, the Golgi apparatus, and lysosomes. The exact structural relationship that exists among the V-ATPases of these intracellular compartments is not currently known. In the present study, we have localized the V-ATPase by light and electron microscopy, using monoclonal antibodies that recognize the V-ATPase present in clathrin-coated vesicles. Localization using light microscopy and fluorescently labeled antibodies reveals that the V-ATPase is concentrated in the juxtanuclear region, where extensive colocalization with the Golgi marker wheat germ agglutinin is observed. The V-ATPase is also present in approximately 60% of endosomes and lysosomes fluorescently labeled using alpha 2-macroglobulin as a marker for the receptor-mediated endocytic pathway. Localization using transmission electron microscopy and colloidal gold-labeled antibodies reveals that the V-ATPase is present at and near the plasma membrane, alone or in association with clathrin. These results provide evidence that the V-ATPases of plasma membrane, endosomes, lysosomes, and the Golgi apparatus are immunologically related to the V-ATPase of clathrin-coated vesicles.
Asunto(s)
Membrana Celular/inmunología , Clatrina/inmunología , Membranas Intracelulares/inmunología , ATPasas de Translocación de Protón/inmunología , Vacuolas/enzimología , Animales , Anticuerpos Monoclonales , Bovinos , Membrana Celular/ultraestructura , Células Cultivadas , Clatrina/ultraestructura , Técnica del Anticuerpo Fluorescente , Aparato de Golgi/inmunología , Aparato de Golgi/ultraestructura , Membranas Intracelulares/ultraestructura , Riñón/citología , Lisosomas/inmunología , Lisosomas/ultraestructura , Microscopía Inmunoelectrónica , Vacuolas/ultraestructuraRESUMEN
Two different types of blood glucose self-monitoring device, designed for use by diabetics (Accu-Chek Advantage* and Accu-Chek III*), were evaluated for the purpose of monitoring glucose concentration in tissue culture media. The Accu-Chek Advantage* meter was found to systematically overestimate the glucose concentration in a variety of commonly used tissue culture media by 50-90%, in comparison with their formulated glucose concentration. The Accu-Chek III* meter reliably estimated glucose concentrations from 100 to 300 mg/dl and overestimated glucose concentrations above 300 mg/dl. The systematic overestimation of glucose concentration in tissue culture fluids by the Accu-Chek Advantage* meter was further investigated. A standard curve was constructed and the meter reading was found to be linearly related to the actual glucose concentration and the best linear fit was given by the formula y = -43.504 + 1.9246x, where y is the meter reading and x is the actual glucose concentration. Rearranging the equation to make x the subject gave the following algorithm x = (y + 43.504) divided by 1.9246 which could be used to correct the 'raw' meter reading. The mean corrected glucose concentrations deviated from the formulated glucose concentration by less than 3.5% in five media tested, indicating that this meter is more than adequate for monitoring glucose consumption by cells growing in hollow fiber cartridge bioreactors, when used in conjunction with this correction factor.
Asunto(s)
Reactores Biológicos , Automonitorización de la Glucosa Sanguínea/instrumentación , Glucosa/metabolismo , Hibridomas/metabolismo , Animales , Reactores Biológicos/normas , Reactores Biológicos/estadística & datos numéricos , Automonitorización de la Glucosa Sanguínea/normas , Automonitorización de la Glucosa Sanguínea/estadística & datos numéricos , Medios de Cultivo/química , Estudios de Evaluación como Asunto , Glucosa/análisis , Humanos , Técnicas Inmunológicas , Ratones , Estándares de ReferenciaRESUMEN
The cellular mechanism of corneal wound contraction after radial keratotomy (RK) was studied in a feline eye model. A total of 10 cat eyes were evaluated at various times from 0-30 days after surgery. Changes in the distribution of intracellular filamentous actin, nonmuscle myosin, alpha-actinin, surface membrane alpha 5 beta 1 integrin, and extracellular fibronectin were studied using immunofluorescence and laser confocal and electron microscopy. From day 3-7, staining for fibronectin increased along the wound margin. By day 7, keratocytes adjacent to the wound margin showed increased f-actin staining with intense staining for fibronectin compared with normal keratocytes. Myosin and alpha 5 beta 1 integrin expression was very weak at this time; alpha-actinin was not found. By day 14, fibroblasts within the wound formed f-actin microfilament bundles (stress fibers) which colocalized with fibronectin. Wound-healing fibroblasts also stained positively for alpha 5 beta 1 integrin, myosin, and alpha-actinin (the latter two were colocalized). The presence of myosin and alpha-actinin in the wound fibroblasts and the re-organization of f-actin into stress fibers by day 14 correlated with the development of wound contraction. A comparison of the cellular distribution of actin, myosin, and alpha-actinin with alpha 5 beta 1 integrin 14 days after injury suggested that integrin was localized along stress fiber bundles during wound contraction. The data from this study suggest that modulation of wound gape during healing of RK wounds may involve transformation of the corneal keratocyte to a myofibroblast-like cell and the subsequent formation of intracellular stress fibers composed of f-actin, nonmuscle myosin, and alpha-actinin. Based on the colocalization of fibronectin filaments and f-actin filaments and the unique distribution of alpha 5 beta 1 integrin, these findings support the hypothesis that the tension within the wound is generated by the formation of intracellular stress fibers and the interactions between stress fibers and the extracellular matrix, mediated by specific membrane receptor molecules.
Asunto(s)
Córnea/fisiopatología , Fibroblastos/fisiología , Queratotomía Radial , Músculo Liso/fisiología , Cicatrización de Heridas , Animales , Gatos , Córnea/ultraestructura , Técnica del Anticuerpo Fluorescente , Microscopía Electrónica , Microscopía Fluorescente , Músculo Liso/patologíaRESUMEN
A perfusion apparatus is employed to reproduce quantifiable pulsatile hemodynamics within freshly excised canine carotid arteries. From measurements of pulsatile intraluminal and transmural pressure and the dynamic radial motion of the vessel wall, calculations are made of the vascular incremental modulus of elasticity and hoop, axial, and radial wall stresses. The results of this investigation suggest that an increase in transmural pressure from 120/80 to 240/120 mmHg produces a marked elevation in incremental modulus and arterial wall stress. These parameters are reduced when transmural pressure is lowered while maintaining intraluminal pressure at physiologic values.
Asunto(s)
Fenómenos Biomecánicos , Arterias Carótidas/fisiología , Perfusión , Animales , Perros , Hemodinámica , Técnicas In Vitro , Rayos Láser , Presión , Flujo PulsátilRESUMEN
The growth of new capillaries from existing vessels (angiogenesis) is of fundamental importance in wound healing and in pathological situations such as proliferative diabetic retinopathy (1), rheumatoid arthritis (2), and tumor growth. Consequently, considerable interest in vascular cell biology has arisen in apparently disparate clinical and experimental fields. Held in common, however, is the hope that an understanding of the cellular and molecular mechanisms that regulate angiogenesis will lead to novel therapeutic agents and targets.