RESUMEN

A novel glass needle-mediated microinjection method for delivery of macromolecules, including proteins and larger transgene DNAs, into the nuclei of blood stem/progenitor cells was developed. Temporary immobilization of cells to extracellular matrix-coated dishes has enabled rapid and consistent injection of macromolecules into nuclei of CD34(+), CD34(+)/CD38(-), and CD34(+)/CD38(-)/Thy-1(lo) human cord blood cells. Immobilization and detachment protocols were identified, which had no adverse effect on cell survival, progenitor cell function (colony forming ability), or stem cell function (NOD/SCID reconstituting ability). Delivery of fluorescent dextrans to stem/progenitor cells was achieved with 52% +/- 8.4% of CD34(+) cells and 42% +/- 14% of CD34(+)/CD38(-)cells still fluorescent 48 hours after injection. Single-cell transfer and culture of injected cells has demonstrated long-term survival and proliferation of CD34(+) and CD34(+)/CD38(-) cells, and retention of the ability of CD34(+)/CD38(-) cells to generate progenitor cells. Delivery of DNA constructs (currently

Asunto(s)

Antígenos CD , Sangre Fetal/citología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Microinyecciones/métodos , Transfección/métodos , Trasplante Heterólogo , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Animales , Antígenos CD34/sangre , Antígenos de Diferenciación/análisis , Adhesión Celular , División Celular , Supervivencia Celular , Ensayo de Unidades Formadoras de Colonias , ADN/genética , Matriz Extracelular/fisiología , Terapia Genética , Vidrio , Proteínas Fluorescentes Verdes , Células Madre Hematopoyéticas/fisiología , Humanos , Recién Nacido , Proteínas Luminiscentes/genética , Glicoproteínas de Membrana , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microinyecciones/instrumentación , NAD+ Nucleosidasa/análisis , Agujas , Fosfoglicerato Quinasa/genética , Proteínas Recombinantes de Fusión/biosíntesis , Antígenos Thy-1/análisis , Transfección/instrumentación

RESUMEN

Over the past decade, significant attention has been devoted to the development of viral vectors (i.e., retrovirus, lentivirus, adeno-associated virus) and conditions capable of transducing hematopoietic stem cells. After several years of disappointing results, recent reports in humans and other primates, most particularly the French report of successful treatment of X-linked severe combined immune deficiency (SCID) [1.], indicate that viral approaches will be successful in treating specific hematopoietic diseases. However, it is clear that alternate non-viral methods of gene delivery and genetic modification offer significant advantages, and may in fact be the only effective approach for treating certain blood diseases. In this review, we focus on glass needle-mediated micro-injection as a method for the delivery of genetic material into blood stem cells, with an emphasis on molecules capable of either compensating gene deletions/mutations or directly repairing gene mutations.

Asunto(s)

Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Animales , ADN/genética , ADN/uso terapéutico , Vectores Genéticos , Humanos , Técnicas In Vitro , Microinyecciones , ARN/genética , ARN/uso terapéutico , Transducción Genética , Virus/genética

RESUMEN

To determine whether pro-inflammatory cytokines modulate intercellular adhesion molecule-1 (ICAM-1; CD54) expression on cultured primary human corneal epithelial cells (HCEs), confluent HCEs were treated with various concentrations of interferon-gamma(IFN-gamma), interleukin-1alpha(IL-1alpha), IL-1beta, IL-4, tumor necrosis factor-alpha (TNF-alpha), or combinations over time. ICAM-1 expression was measured by flow cytometry and/or a cell-based ELISA using a monoclonal mouse anti-human CD54 antibody. The apparent MW of ICAM-1 protein was determined by immunoprecipitation of biotinylated HCEs. RT-PCR was used to detect ICAM-1 RNA. The mature cell surface form of HCE ICAM-1 was approximately 110 kDa as determined by immunoprecipitation. IFN-gammaand TNF-alpha induced both dose- and time-dependent increases in ICAM-1 expression. An approximately 20-fold increase in ICAM-1 was seen at 50-100 U IFN-gamma ml-1. ICAM-1 specific mRNA accumulated approximately 4.5-fold after IFN-gammatreatment. TNF-alpha(100 U ml-1) induced a consistent approximately 6.0-fold increase in ICAM-1 expression. When IFN-gammaand TNF-alpha were mixed, at sub-optimal concentrations of each, a synergistic effect on ICAM-1 expression was not detected. Neither IL-4, IL-1alpha nor IL-1beta affected ICAM-1 expression in a consistent fashion. In summary, ICAM-1 was modulated on primary human corneal epithelial cells by the cytokines IFN-gamma and TNF-alpha in a dose- and time-dependent fashion. Cytokine modulation of corneal epithelial cell ICAM-1 during inflammation may contribute to corneal epithelial cell injury by aiding the attachment of inflammatory cells such as eosinophils which express the receptor for ICAM-1, the beta2 integrins (CD11a,b,c/CD18).

Asunto(s)

Citocinas/farmacología , Epitelio Corneal/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Técnicas de Cultivo de Célula , Ensayo de Inmunoadsorción Enzimática , Epitelio Corneal/citología , Citometría de Flujo , Humanos , Molécula 1 de Adhesión Intercelular/genética , Interferón gamma/farmacología , Interleucinas/farmacología , Pruebas de Precipitina , ARN Mensajero/genética , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

The rat liver sinusoidal endothelial cell (LEC) hyaluronan (HA) receptor was previously identified using a photoaffinity HA derivative (J. Biol. Chem., 267, 20451-20456, 1992). Two polypeptides with M(r) = 175,000 and 166,000, were consistently crosslinked, suggesting that the LEC HA receptor is an oligomer. Whether one or both subunits participate in HA binding, was not determined. Here we investigate the HA-subunit interactions and the potential oligomeric nature of the LEC HA receptor. When Sephacryl-400 gel filtration chromatography was used to enrich the HA receptor, the 175 kDa polypeptide was the major band seen by SDS-PAGE analysis. Little staining was seen at 166 kDa, suggesting that the 175 kDa protein could be separated from the 166 kDa protein and still retain HA-binding activity. A ligand blot assay was used to determine if each individual subunit could bind HA. LEC proteins were separated by nonreducing SDS-PAGE, and then immobilized onto nitrocellulose. 125I-HA bound to a 175 kDa polypeptide but not to the 166 kDa protein. A high molecular weight band of approximately 300,000 also bound 125I-HA. 125I-HA binding to the 175 and 300 kDa proteins showed the same specificity of competition with a panel of carbohydrates as the bona fide LEC HA receptor. The 175 kDa HA-binding subunit may be nonglobular (asymmetric), since its apparent size by SDS-PAGE is dependent on the polyacrylamide gel pore size; M(r) increases as porosity decreases. LECs were crosslinked to an 125I-labeled photoaffinity HA derivative and the HA saccharides were then released with hyaluronidase. After SDS-PAGE without reduction, radiolabeled bands were seen at 175 and 166 kDa (3:1 ratio), and a high MW (approximately 300,000) species was also detected. These data support an oligomeric model of the LEC HA receptor, and show that the 175 kDa protein possesses HA-binding activity independent from the 166 kDa polypeptide.

Asunto(s)

Receptores de Hialuranos/metabolismo , Hígado/metabolismo , Animales , Western Blotting , Células Cultivadas , Endotelio/citología , Endotelio/metabolismo , Radioisótopos de Yodo , Ligandos , Hígado/citología , Masculino , Proteínas de la Membrana/aislamiento & purificación , Unión Proteica , Ratas , Ratas Sprague-Dawley

RESUMEN

Recent reports have demonstrated the expression of intercellular adhesion molecule-1 (ICAM-1; CD54) on the epithelium in various allergic diseases and inflammatory conditions, including the bronchial epithelium of patients with allergic asthma, conjunctival epithelium of allergic patients after allergen-specific challenge, and corneal epithelium of rejected corneal allografts. We investigated the presence of ICAM-1 expression on the corneal epithelium from a patient with vernal keratoconjunctivitis (VKC). Immunohistochemical staining of the diseased cornea demonstrated abundant ICAM-1 expression on the corneal epithelium. Immunoreactive ICAM-1 appeared to localize primarily to the cells of the basal and middle layers of the corneal epithelium. No staining was detected on the ocular surface epithelium. The normal, healthy cornea demonstrated no significant ICAM-1 expression on any of the epithelial layers, similar to that previously reported. To the best of our knowledge, this is the first report of ICAM-1 expression on the corneal epithelium from a patient with VKC.

Asunto(s)

Conjuntivitis Alérgica/metabolismo , Córnea/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Adulto , Sitios de Unión , Conjuntivitis Alérgica/patología , Córnea/patología , Epitelio/metabolismo , Epitelio/patología , Humanos , Inmunohistoquímica , Masculino , Linaje

RESUMEN

Rat liver endothelial cells (LECs) express a membrane-associated Ca2+-dependent hyaluronan-binding activity (CaHA-BP) which is distinct from the Ca2+-independent, endocytic LEC HA receptor (Yannariello-Brown et al., J. Cell Biochem., 48, 73-80, 1992). The CaHA-BP is specific for a subset of glycosaminoglycans, since Ca2+-dependent binding of 125I-HA (approximately 80 kDa) to LECs was competed with a 100-fold excess (w/w) of HA, chondroitin sulfate, and heparin, but not with chondroitin. The CaHA-BP activity on intact LECs was pH-dependent. Optimal binding occurred at pH 6.0; no binding was detected at pH values or= 9. 125I-HA, pre-bound in the presence of Ca2+, could also be dissociated with an acidic buffer (pH 5.0), as well as the divalent cation chelators EDTA and EGTA. 125I-HA binding was stimulated by divalent cations other than Ca2+, such as Mg2+, Mn2+, and Ba2+; with the exception of Zn2+. A photoaffinity crosslinking reagent (125I-ASD-HA) was used to identify specifically crosslinked polypeptides on LECs. In the absence of Ca2+, and in the presence of EGTA, only bands at 175/166 kDa were consistently crosslinked. These bands have been previously identified as the LEC Ca2+-independent endocytic HA receptor (Yannariello-Brown et al., J. Biol. Chem., 267, 20451-20455, 1992). In the presence of Ca2+, crosslinking was consistently seen to a 68 kDa polypeptide. Crosslinking was competed with a 100-fold excess (w/w) of HA. These and other data suggest that a 68 kDa protein is the most likely candidate for the CaHA-BP in LECs.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Endotelio/metabolismo , Ácido Hialurónico/metabolismo , Hígado/metabolismo , Animales , Azidas , Unión Competitiva , Calcio/metabolismo , Cationes Bivalentes , Células Cultivadas , Reactivos de Enlaces Cruzados , Medios de Cultivo , Endotelio/citología , Humanos , Radioisótopos de Yodo , Hígado/citología , Fotoquímica , Ratas , Succinimidas

RESUMEN

We have developed a sensitive ligand blot assay to detect hyaluronan (HA) binding proteins in cell extracts using 125I-HA. Samples to be tested are electrophoresed using standard, nonreducing SDS-PAGE conditions, electro-transferred to nitrocellulose then blocked in buffer containing Tween 20. After incubation with 125I-HA the nitrocellulose is washed and HA-binding proteins are detected by autoradiography. This method was used to detect different HA-binding proteins in isolated rat liver cell preparations. Two HA-binding bands of 175 kD and 350 kD were detected in sinusoidal endothelial cells. Both bands were competed virtually 100% with nonlabeled HA. Using rat hepatocytes, the assay detected major bands at 85 kD and 180 kD. In addition, histones present in both cell types were readily detected in the low MW region. Thus, two different liver cell types show different HA-binding patterns. The blocking procedure is critical for successful renaturation of HA-binding activity, since substitution of BSA for Tween 20 did not result in detectable 125I-HA-binding. This ligand blot assay will be a powerful tool to detect HA-binding proteins in various other tissues and cell types.

Asunto(s)

Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Hígado/citología , Hígado/metabolismo , Animales , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Endotelio/citología , Endotelio/metabolismo , Receptores de Hialuranos/análisis , Receptores de Hialuranos/aislamiento & purificación , Radioisótopos de Yodo , Peso Molecular , Polisorbatos , Ratas

RESUMEN

Circulating hyaluronan (HA) levels were investigated as a function of age and diet in Fischer 344 male rats. A biphasic pattern of age-related changes was observed in rats fed ad libitum a diet in which the protein source was soya/fish meal. HA levels in 3- to 6- and 22- to 29-mo-old rats were not statistically different. However, HA levels in 12- to 20-mo-old rats were 10-29% of the levels in younger or aged adults. HA levels were also measured in rats fed ad libitum a semisynthetic diet in which the protein source was hydrolyzed casein. Whereas the two colonies exhibited similar biphasic age-related changes, HA levels differed 4- to 20-fold at every age examined. Caloric restriction affected HA levels in 19-mo-old casein-fed rats; HA levels were 2.3 times higher than age-matched controls and were not statistically different from young or aged animals. Serum and plasma HA levels were identical in the same individuals at all ages tested. These data suggest that HA turnover and metabolism in the rat are affected by age, dietary composition, and caloric intake.

Asunto(s)

Envejecimiento/sangre , Cricetinae/sangre , Dieta , Ácido Hialurónico/sangre , Ratas/sangre , Animales , Masculino , Mesocricetus , Plasma , Ratas Endogámicas F344 , Ratas Sprague-Dawley

RESUMEN

The release and intracellular accumulation of 125I-hyaluronan degradation products was studied in cultured liver endothelial cells with hyaluronan oligosaccharides (relative molecular mass = approximately 44,000) uniquely modified and radiolabeled at the terminal reducing sugar. Two methods were combined to measure 125I-hyaluronan degradation by liver endothelial cells. (a) Cetylpyridinium chloride precipitation of hyaluronan oligosaccharides was used as a rapid, convenient assay to monitor the appearance of hyaluronan degradation products. Hyaluronan oligosaccharides less than 54 to 60 monosaccharides in length were not precipitated with cetylpyridinium chloride and thus were assessed as degraded. (b) Gel filtration chromatography was used to estimate the size range of oligosaccharides produced by liver endothelial cells. After internalization of 125I-hyaluronan, liver endothelial cells released radioactive degradation products into the culture media after a lag period of 2.5 to 3.0 hr. The intracellular accumulation of degraded 125I-hyaluronan was linear for at least 2 hr even though no degradation products were released. The long lag before release of degraded 125I-hyaluronan is likely caused by the modified chemical structure at the reducing end of the hyaluronan derivative; the derivative acts like a residualizing label. After this lag the release of degraded 125I-hyaluronan proceeded linearly for up to 12 hr. The extracellular 125I-hyaluronan degradation products eluted with a distribution coefficient of 1.3 on a gel filtration column. The major intracellular 125I-labeled degradation product showed the same retardation (distribution coefficient = 1.3). This retention may be caused by the hydrophobic aromatic and alkyl modifications to the former reducing sugar, also characteristics of a residualizing label. In addition, at least two larger minor intermediates were observed intracellularly. The rate of intracellular 125I-hyaluronan degradation was dependent on hyaluronan concentration and reached a maximal rate (159 molecules/cell/sec) at 2 x 10(-7) mol/L. This was about half the maximal rate of endocytosis (285 molecules/cell/sec) at a hyaluronan concentration of 1.3 x 10(-7) mol/L. The apparent ligand concentration that gives half-maximal responses for endocytosis and intracellular degradation was 0.6 x 10(-7) and 1.0 x 10(-7) mol/L, respectively.

Asunto(s)

Ácido Hialurónico/metabolismo , Hígado/metabolismo , Animales , Células Cultivadas , Cetilpiridinio , Precipitación Química , Endocitosis , Endotelio/citología , Endotelio/metabolismo , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/farmacocinética , Hígado/citología , Peso Molecular , Oligosacáridos/metabolismo , Ratas

RESUMEN

The Ca(2+)-independent endocytic hyaluronan (HA) receptor in rat liver sinusoidal endothelial cells (LECs) was identified using a novel cross-linking derivative of HA. The heterobifunctional, photoactivatable, reducible reagent sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) was coupled to the terminal amino group of uniquely modified HA-amine oligosaccharides (M(r) approximately 60,000) and subsequently iodinated. 125I-ASD-HA bound to cultured LECs with similar specificity and affinity as a previously characterized 125I-HA-amine/Bolton-Hunter adduct. Permeabilized LECs were incubated with 125I-ASD-HA with 10 mM EGTA and photolysed with UV light. Detergent extracts were reduced to release the HA oligosaccharides and radiolabeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Two polypeptides were consistently and equally labeled at M(r) = 175,000 and 166,000. Photoaffinity labeling of these two proteins was virtually identical in cultured LECs or membranes and was competed greater than 90% with a 100-fold excess of HA. As with the previously characterized bona fide LEC HA receptor, cross-linking was also competed by chondroitin sulfate and heparin, but less efficiently by chondroitin and not with galacturonan. We conclude that the Ca(2+)-independent LEC HA receptor is composed of at least two polypeptides of M(r) approximately 175,000 and 166,000 and may exist as a heterodimer of M(r) approximately 340,000. We also conclude that the LEC HA receptor is distinct from the CD44 family of HA-binding proteins.

Asunto(s)

Azidas , Reactivos de Enlaces Cruzados , Endocitosis , Ácido Hialurónico , Hígado/química , Receptores de Superficie Celular/análisis , Succinimidas , Animales , Autorradiografía , Calcio/fisiología , Cationes Bivalentes , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Endotelio/química , Receptores de Hialuranos , Hígado/citología , Masculino , Fotoquímica , Ratas , Ratas Sprague-Dawley

RESUMEN

Isolated and cultured rat liver sinusoidal endothelial cells (LECs) retain the ability to specifically bind 125I-hyaluronan (HA) and internalize it using a coated pit pathway [Biochem J, 257:875-884, 1989]. Here we have determined the effect of Ca+2 on the binding and endocytosis of HA by LECs. 125I-HA binding to intact LECs at 4 degrees C occurred both in the absence (10 mM EGTA) or the presence of physiologic concentrations of Ca+2 (1.8 mM). However, the specific binding of 125I-HA to LECs increased linearly with increasing Ca+2 concentrations. After permeabilization with the nonionic detergent digitonin, the Ca(+2)-independent HA binding activity increased approximately 743%, while the Ca(+2)-dependent binding activity was enhanced only approximately 46%. Therefore, the Ca(+2)-dependent HA binding activity appears not to be intracellular, whereas the Ca(+2)-independent HA receptor is found both inside LECs and on the cell surface. When LECs were allowed to endocytose 125I-HA at 37 degrees C in 10 mM EGTA or in 1.8 mM Ca+2, no differences were seen in the extent or rate of endocytosis. When LECs were allowed to endocytose 125I-HA in the presence of 10 mM Ca+2, the amount of cell-associated radioactivity increased approximately 20-50-fold. However, this additional cell-associated 125I-HA was not sensitive to hyperosmolarity and was removed by washing the cells in 10 mM EGTA at 4 degrees C. Therefore, the Ca(+2)-dependent cell-associated 125I-HA had accumulated on the cell surface and had not been internalized. From these studies we conclude that LECs have at least two types of specific HA binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Calcio/farmacología , Endocitosis/efectos de los fármacos , Endotelio/metabolismo , Ácido Hialurónico/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Permeabilidad de la Membrana Celular , Células Cultivadas , Endotelio/citología , Receptores de Hialuranos , Hígado/citología , Masculino , Concentración Osmolar , Ratas , Ratas Endogámicas

RESUMEN

Rat liver sinusoidal endothelial cells (LECs) mediate the removal of hyaluronan (HA) from the circulation via a specific Ca(2+)-independent endocytic receptor. To characterize the receptor biochemically, detergent-soluble extracts were prepared from crude LEC membranes. Using a dot blot assay to quantitate 125I-HA binding activity in CHAPS-solubilized membranes, we detected not only specific Ca(2+)-independent but also specific Ca(2+)-dependent HA-binding activity. Both HA-binding activities behave as integral membrane-associated proteins; they are not released from LEC membranes by treatment at pH 11, and they require detergent for extraction. The Ca(2+)-independent HA receptor was inactivated by treatment at 56 degrees C for 30 min or with 200 mM DTT at 4 degrees C for 30 min, whereas the Ca(2+)-dependent activity actually increased by 75% after treatment at 56 degrees C and only 20% of the Ca(2+)-dependent activity was lost after DTT treatment. A two-cycle membrane extraction protocol using CHAPS partially separated the two HA-binding activities. Eight millimolar KCl and 0.5% CHAPS extracted approximately 50% of the Ca(2+)-independent HA receptor, but only 4-11% of the Ca(2+)-dependent activity. When the KCl and CHAPS concentrations were increased to 2.0 M and 1.5%, respectively, the remaining HA receptor, as well as 89-96% of the Ca(2+)-dependent activity, was then extracted. The Ca(2+)-independent and Ca(2+)-dependent activities could also be further separated using Sephacryl S-400 gel filtration chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Calcio/farmacología , Proteínas Portadoras/análisis , Ácidos Cólicos , Endocitosis , Endotelio/química , Ácido Hialurónico/metabolismo , Hígado/química , Receptores de Superficie Celular/análisis , Animales , Carbonatos/farmacología , Cromatografía en Gel , Humanos , Receptores de Hialuranos , Concentración de Iones de Hidrógeno , Masculino , Unión Proteica , Ratas , Ratas Endogámicas , Receptores de Superficie Celular/química , Receptores de Superficie Celular/efectos de los fármacos , Solubilidad , Temperatura

RESUMEN

We have identified collagen-binding proteins in detergent extracts of metabolically labelled bovine aortic endothelial cells (BAEC) by collagen type IV-Sepharose affinity chromatography. The major collagen type IV-binding protein identified by SDS/PAGE had a molecular mass of 48 kDa, which we term the 'collagen-binding 48 kDa protein' (CB48). The pI of CB48 was 8.0-8.3 in a two-dimensional gel system, running non-equilibrium pH gel electrophoresis in the first dimension and SDS/PAGE in the second dimension. Under these conditions CB48 separated into two major (a and b) and one minor isoform (c); a was the most basic of the three isoforms. Two-dimensional chymotryptic peptide maps derived from each individual isoform were virtually identical. The charge differences between the isoforms were due in part to differential H3(32)PO4 incorporation by the protein. CB48 bound to intact collagen type IV and the collagenous region of collagen type IV, but not to the globular NC1 domain. Cell-surface labelling and indirect immunofluorescence experiments localized the bulk of CB48 intracellularly in the endoplasmic reticulum Golgi region, with a minor population of molecules on the cell surface. A specific rabbit polyclonal anti-CB48 serum did not inhibit the attachment or spreading of BAEC to collagen type IV in an 'in vitro' adhesion assay, suggesting that the cell-surface population of CB48 is not involved in BAEC adhesion. We conclude that CB48 is a collagen-binding phosphoprotein that interacts with the collagenous domain of collagen type IV and may be involved in intracellular transport of collagen molecules.

Asunto(s)

Colágeno/metabolismo , Endotelio Vascular/metabolismo , Fosfoproteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Aorta , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Cromatografía de Afinidad , Quimotripsina , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Sueros Inmunes , Peso Molecular , Mapeo Peptídico , Fosfoproteínas/análisis , Fosfoproteínas/aislamiento & purificación , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/aislamiento & purificación , Receptores de Colágeno

RESUMEN

The vessel wall is composed of heterogeneous cell populations residing in a variety of vascular beds. Each cell type has different functions and morphologies but all of them have a role in the repair process following vascular injury. Responses to injury vary depending upon the type and extent of the injury and the vascular bed affected. The sheet migration and proliferation exhibited by large vessel endothelial cells is in striking contrast to the migration through soft tissues and tube formation exhibited by microvascular endothelial cells in response to injury. Vascular smooth muscle cells respond to injury by migrating into the intima, proliferating and synthesizing matrix, causing intimal thickening. The response to injury by vascular cells appears to be modulated, in part, by the composition and organization of the surrounding matrix and the various platelet factors and cytokines found at sites of injury. Furthermore, evidence has been accrued in culture, suggesting that solid phase (matrix) and soluble factors modulate each other's effects on local vascular cell populations following injury.

Asunto(s)

Vasos Sanguíneos/citología , Músculo Liso Vascular/fisiología , Animales , Humanos , Músculo Liso Vascular/citología

RESUMEN

Current hypotheses suggest that the extracellular matrix modulates cellular function in physiologic homeostasis and during injury and repair and that selected cellular functions are cell and nuclear size dependent. For elucidation of the role of extracellular matrix-driven cell size changes in the modulation of endothelial cell proliferation and sheet migration an in vitro model system was used that allows for the culture of bovine aortic endothelial cells (BAEC) on various purified extracellular matrix components. BAEC exhibited distinct patterns in rates of attachment, spreading, migration, and proliferation when cultured on the selected extracellular matrix components laminin, types I and III collagen, type IV collagen, and fibronectin. In addition, there was a correlation between cell and nuclear size (on the various matrices tested) and rates of cell attachment and spreading. In contrast, an inverse correlation was noted between cell and nuclear size (on the various matrices tested) and proliferation and sheet migration. These data demonstrate that endothelial cells respond to matrix components in specific but complex fashions, mediated, in part, by changes in cell and nuclear size.

Asunto(s)

Aorta/citología , Endotelio Vascular/citología , Matriz Extracelular/fisiología , Animales , Aorta/ultraestructura , Adhesión Celular , División Celular , Movimiento Celular , Endotelio Vascular/ultraestructura

RESUMEN

Affinity chromatography and immunolocalization techniques were used to investigate the mechanism(s) by which endothelial cells interact with the basement membrane component laminin. Bovine aortic endothelial cells (BAEC) membranes were solubilized and incubated with a laminin-Sepharose affinity column. SDS-PAGE analysis of the eluted proteins identified a 69-kD band as the major binding protein, along with minor components migrating at 125, 110, 92, 85, 75, 55, and 30 kD. Polyclonal antibodies directed against a peptide sequence of the 69-kD laminin-binding protein isolated from human tumor cells identified this protein in BAEC lysates. In frozen sections, these polyclonal antibodies and monoclonal antibodies raised against human tumor 69-kD stained the endothelium of bovine aorta and the medial smooth muscle cells, but not surrounding connective tissue or elastin fibers. When nonpermeabilized BAEC were stained in an in vitro migration assay, there appeared to be apical patches of 69 kD staining in stationary cells. However, when released from contact inhibition, 69 kD was localized to ruffling membranes on cells at the migrating front. Permeabilized BAEC stained for 69 kD diffusely, with a granular perinuclear distribution and in linear arrays throughout the cell. During migration a redistribution from diffuse to predominanately linear arrays that co-distributed with actin microfilaments was noted in double-label experiments. The 69-kD laminin-binding protein colocalized with actin filaments in permeabilized cultured microvascular endothelial cells in a continuous staining pattern at 6 h postplating which redistributed to punctate patches along the length of the filaments at confluence (96 h). In addition, 69 kD co-distribution with laminin could also be demonstrated in cultured subconfluent cells actively synthesizing matrix. Endothelial cells express a 69-kD laminin-binding protein that is membrane associated and appears to colocalize with actin microfilaments. The topological distribution of 69 kD and its cytoskeletal associations can be modulated by the cell during cell migration and growth suggesting that 69 kD may be a candidate for a membrane protein involved in signal transduction from extracellular matrix to cell via cytoskeletal connections.

Asunto(s)

Endotelio Vascular/análisis , Laminina/metabolismo , Receptores Inmunológicos/análisis , Citoesqueleto de Actina/análisis , Citoesqueleto de Actina/metabolismo , Animales , Aorta , Bovinos , Adhesión Celular , Movimiento Celular , Células Cultivadas , Cromatografía de Afinidad , Endotelio Vascular/citología , Matriz Extracelular/análisis , Matriz Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoensayo , Masculino , Microcirculación , Ratas , Ratas Endogámicas , Receptores de Laminina