RESUMO
In this review we discuss features of mRNA synthesis and modifications used to minimize immune response and prolong efficiency of the translation process in vivo. Considerable attention is given to the use of liposomes and nanoparticles containing lipids and polymers for the mRNA delivery. Finally we briefly discuss mRNAs which are currently in the clinical trials for cancer immunotherapy, vaccination against infectious diseases, and replacement therapy.
Assuntos
Portadores de Fármacos/química , RNA Mensageiro/metabolismo , Animais , Humanos , Imunidade Inata , Imunoterapia , Lipossomos/química , Nanopartículas/química , Neoplasias/imunologia , Neoplasias/terapia , RNA Mensageiro/química , RNA Mensageiro/genética , VacinaçãoRESUMO
In the vertebrate embryo, neural cell types are organized spatially along the dorsoventral axis of the neural tube and differ by expression of cell-intrinsic determinants and by their adhesive and locomotory properties. Thus, dorsally, neural crest cells (NCC) show a strong propensity to disperse and migrate, whereas cells situated ventrally are highly cohesive and poorly motile. Members of the bone morphogenetic proteins have been shown to exert a dual role in the specification of dorsal neuroepithelial cells and in the dispersion of NCCs. To test whether Sonic hedgehog (Shh), another signaling molecule involved in the patterning of the ventral neural tube, might also contribute to the control of the adhesive and migratory potential of neuroepithelial cells, we analyzed the effect of ectopic Shh on NCC dispersion from neural tube explants cultured in vitro. The addition of Shh to the migration substrate of NCC caused inhibition of their dispersion. The effect of Shh on cell migration was reversible and was not accounted for by alterations of the specification, delamination, proliferation, and survival of NCCs but could be essentially attributed to a decreased cell-substrate adhesion mediated by integrins. In addition, Shh activity on cell migration was mediated by a specific N-terminal region of the molecule and was independent from the signaling cascade elicited by the Patched-Smoothened receptor and involving the Gli transcription factors. Our study therefore reveals an unanticipated role for Shh in regulating adhesion and migration of neuroepithelial cells that is discernable from its inductive, mitogenic, and trophic functions.
Assuntos
Crista Neural/citologia , Proteínas Oncogênicas/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/fisiologia , Adesão Celular , Movimento Celular , Embrião de Galinha , Proteínas Hedgehog , Humanos , Integrinas/fisiologia , Codorniz , Receptor Smoothened , Proteína GLI1 em Dedos de ZincoRESUMO
We have shown that the integrin alphavbeta6 activates latent TGF-beta in the lungs and skin. We show here that mice lacking this integrin are completely protected from pulmonary edema in a model of bleomycin-induced acute lung injury (ALI). Pharmacologic inhibition of TGF-beta also protected wild-type mice from pulmonary edema induced by bleomycin or Escherichia coli endotoxin. TGF-beta directly increased alveolar epithelial permeability in vitro by a mechanism that involved depletion of intracellular glutathione. These data suggest that integrin-mediated local activation of TGF-beta is critical to the development of pulmonary edema in ALI and that blocking TGF-beta or its activation could be effective treatments for this currently untreatable disorder.
Assuntos
Antígenos de Neoplasias , Síndrome do Desconforto Respiratório/etiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Bleomicina , Barreira Alveolocapilar/fisiologia , Células Cultivadas , Endotoxinas , Glutationa/metabolismo , Integrinas/genética , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases , Alvéolos Pulmonares/metabolismo , Edema Pulmonar/etiologia , Edema Pulmonar/patologia , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/administração & dosagem , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
BACKGROUND & AIMS: Transforming growth factor (TGF)-beta has been implicated in many fibrotic conditions. However, its mechanistic role in radiation toxicity is equivocal despite compelling correlative evidence. This study assessed whether in vivo administration of a soluble TGF-beta type II receptor (TbetaR-II) protein ameliorates intestinal radiation injury (radiation enteropathy). METHODS: A recombinant fusion protein, consisting of the extracellular portion of mouse TbetaR-II and the Fc portion of mouse immunoglobulin (Ig) G, was produced. A 5-cm segment of mouse ileum was exposed to 19 Gy x-radiation. TbetaR-II:Fc fusion protein (1 mg/kg every other day) or mouse IgG was administered from 2 days before to 6 weeks after irradiation. Radiation injury was assessed at 6 weeks using quantitative histology, morphometry, and immunohistochemistry. Collagen was measured colorimetrically, and TGF-beta1 messenger RNA was assessed with fluorogenic probe reverse-transcription polymerase chain reaction. RESULTS: Compared with IgG controls, TbetaR-II:Fc-treated mice exhibited less structural injury, preservation of mucosal surface area, and less intestinal wall fibrosis. Intestinal TGF-beta1 messenger RNA increased in TbetaR-II:Fc-treated mice, whereas TGF-beta immunoreactivity decreased. TbetaR-II:Fc treatment increased crypt cell proliferation but otherwise did not affect unirradiated intestine. CONCLUSIONS: Long-term modulation of TGF-beta with a TbetaR-II:Fc fusion protein is feasible and ameliorates radiation enteropathy. These data confirm the putative role of TGF-beta in intestinal radiation fibrosis.
Assuntos
Enteropatias/tratamento farmacológico , Lesões por Radiação/tratamento farmacológico , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Células CHO , Colágeno/metabolismo , Cricetinae , Íleo/efeitos dos fármacos , Íleo/metabolismo , Íleo/patologia , Íleo/efeitos da radiação , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Enteropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Lesões por Radiação/patologia , Receptores de Fatores de Crescimento Transformadores beta/química , Solubilidade , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1RESUMO
In the present study, we investigated the role of the CD40L-CD40 pathway in a model of progressive atherosclerosis. ApoE-/- mice were treated with an anti-CD40L antibody or a control antibody for 12 wk. Antibody treatment started early (age 5 wk) or was delayed until after the establishment of atherosclerosis (age 17 wk). In both the early and delayed treatment groups, anti-CD40L antibody did not decrease plaque area or inhibit lesion initiation or age-related increase in lesion area. The morphology of initial lesions was not affected, except for a decrease in T-lymphocyte content. Effects of anti-CD40L antibody treatment on the morphology of advanced lesions were pronounced. In both the early and delayed treatment groups, T-lymphocyte content was significantly decreased. Furthermore, a pronounced increase in collagen content, vascular smooth muscle cell/myofibroblast content, and fibrous cap thickness was observed. In the delayed treatment group, a decrease in lipid core and macrophage content occurred. Interestingly, advanced lesions of anti-CD40L antibody-treated mice exhibited an increased transforming growth factor beta1 immunoreactivity, especially in macrophages. In conclusion, both early and delayed treatment with an anti-CD40L antibody do not affect atherosclerotic lesion initiation but do result in the development of a lipid-poor collagen-rich stable plaque phenotype. Furthermore, delayed treatment with anti-CD40L antibody can transform the lesion profile from a lipid-rich to a lipid-poor collagen-rich phenotype. Postulated mechanisms of this effect on plaque phenotype are the down-regulation of proinflammatory pathways and up-regulation of collagen-promoting factors like transforming growth factor beta.
Assuntos
Apolipoproteínas E/deficiência , Arteriosclerose , Antígenos CD40/imunologia , Glicoproteínas de Membrana/imunologia , Fatores Etários , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Apolipoproteínas E/genética , Arteriosclerose/genética , Arteriosclerose/imunologia , Ligante de CD40 , Modelos Animais de Doenças , Camundongos , Linfócitos T/imunologiaRESUMO
Atherosclerosis is a systemic disease of the large arteries, and activation of inflammatory pathways is important in its pathogenesis. Increasing evidence supports the importance of CD40-CD154 interactions in atherosclerosis, interactions originally known to be essential in major immune reactions and autoimmune diseases. CD40 is present on atheroma-derived cells in vitro and in human atheromata in situ. Ligation of CD40 on atheroma-associated cells in vitro activates the production of chemokines, cytokines, matrix metalloproteinases, adhesion molecules and tissue factor, substances responsible for lesion progression and plaque destabilization. Administration of antibody against CD154 to low-density lipoprotein receptor-deficient mice has been shown to reduce atherosclerosis and decrease T-lymphocyte and macrophage content; however, only initial lesions were studied. Here, we determined the effect of genetic disruption of CD154 in ApoE-/- mice in both initial and advanced atherosclerotic lesions. Plaque area was reduced 550%. In contrast to previous reports, initial lesion development was not affected. Advanced plaques in CD154-/-ApoE-/- mice had a less-lipid-containing, collagen-rich, stable plaque phenotype, with a reduced T-lymphocyte/macrophage content. These data indicate that CD40-CD154 signaling is important in late atherosclerotic changes, such as lipid core formation and plaque destabilization.
Assuntos
Arteriosclerose/imunologia , Glicoproteínas de Membrana/imunologia , Animais , Sequência de Bases , Ligante de CD40 , Colesterol/sangue , Primers do DNA , Progressão da Doença , Humanos , Imuno-Histoquímica , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/imunologia , Triglicerídeos/sangueRESUMO
BACKGROUND: Transforming growth factor beta (TGF-beta) is a key mediator of collagen synthesis in the development of lung fibrosis. It has previously been shown that the administration of TGF-beta antibody and TGF-beta binding proteoglycan, decorin, reduced bleomycin (BL) induced lung fibrosis in animals. The present study was carried out to investigate whether intratracheal instillation of TGF-beta soluble receptor (TR) would minimise the BL induced lung fibrosis in hamsters. METHODS: The effect of a recombinant TR (TGFbetaRII) on the lung collagen accumulation was evaluated in a BL hamster model of pulmonary fibrosis. Animals were divided into four groups and intratracheally injected with saline or BL at 6.5 U/4 ml/kg followed by intratracheal instillation of phosphate buffered saline (PBS) or 4 nmol TR in 0.3 ml twice a week. Twenty days after the first intratracheal instillation the hamsters were killed for bronchoalveolar lavage (BAL) fluid, biochemical, and histopathological analyses. RESULTS: Treatment of hamsters with TR after intratracheal instillation of BL significantly reduced BL induced lung fibrosis as shown by decreases in the lung hydroxyproline level and prolyl hydroxylase activity, although they were still significantly higher than those of the saline control. Histopathological examination showed a considerable decrease in BL induced fibrotic lesions by TR treatment. However, TR did not prevent the BL induced increases in total cells and protein in the BAL fluid. CONCLUSIONS: These results suggest that TR has antifibrotic potential in vivo and may be useful in the treatment of fibrotic diseases where increased TGF-beta is associated with excess collagen accumulation.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Bleomicina/efeitos adversos , Fibrose Pulmonar/prevenção & controle , Receptores de Fatores de Crescimento Transformadores beta/uso terapêutico , Animais , Líquido da Lavagem Broncoalveolar , Divisão Celular/efeitos dos fármacos , Colágeno , Cricetinae , Avaliação Pré-Clínica de Medicamentos , Hidroxiprolina/farmacologia , Masculino , Mesocricetus , Oxigenases de Função Mista/farmacologia , Fibrose Pulmonar/induzido quimicamente , Receptores de Fatores de Crescimento Transformadores beta/administração & dosagemRESUMO
Transcriptional regulation of smooth muscle cell (SMC) differentiation is a rapidly growing area of interest that has relevance for understanding intimal disease. Despite the wealth of data accumulating in vitro, however, no study has compared the cell-specific marker profile, transfectability, promoter activity, and growth characteristics among several SMC culture systems. Accordingly, we performed a comprehensive analysis of the marker profile, growth properties, transfectability, and SMC promoter activity in four rat SMC lines (A7r5, adult and pup aortic, and PAC1). Despite alterations in chromosomal number and structure, A7r5, adult aortic, and PAC1 cells express all SMC markers studied including SM alpha-actin, SM calponin, SM22, tropoelastin, and to a lesser extent, SM myosin heavy chain (SMMHC). In contrast, pup aortic cells express very low or undetectable levels of all the above markers except tropoelastin. Adult aortic, pup, and PAC1 cells display similar growth curves and levels of proto-oncogene transcripts, whereas those in the A7r5 line are comparatively less. All cell lines studied except pup cells show expression of SMC differentiation genes during active growth, indicating that growth and differentiation are not mutually exclusive in cultured smooth muscle. Transfection studies reveal dramatic differences in DNA uptake and SMC-restricted promoter activity between cell lines. Collectively, these results provide detailed information relating to SMC molecular biology in culture that should facilitate the selection of a cell line for studying the transcriptional regulatory mechanisms underlying SMC differentiation.
Assuntos
Músculo Liso Vascular/metabolismo , Actinas/análise , Animais , Biomarcadores , Proteínas de Ligação ao Cálcio/análise , Divisão Celular , Linhagem Celular , Proteínas dos Microfilamentos , Músculo Liso Vascular/citologia , Cadeias Pesadas de Miosina/análise , Regiões Promotoras Genéticas , Ratos , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Soroalbumina Bovina/farmacologia , Transfecção , CalponinasRESUMO
Differentiation of vascular smooth muscle cells (SMCs) is characterized by several molecular transitions. As differentiation proceeds, proteins of the cytoskeletal and contractile apparatus, such as alpha-smooth muscle actin, smooth muscle myosin, calponin, and heavy caldesmon, and the expression of the membrane-related protein smooth muscle phosphoglucomutase-related protein increase, whereas the expression of other proteins, such as fibronectin splice variants with extradomains A (EDA) and B (EDB), decreases. In this study, we investigated the differentiation of the SMCs of the ductus arteriosus during the development of intimal thickening. Ascending and descending aortas of the same age were used for comparison because these vessels lack intimal thickening. In the fetal ductus arteriosus, a relatively early differentiation of the contractile apparatus was observed compared with the ascending and descending aortas. EDA and EDB expression was already low, being similar in the ductus and descending aorta and even lower in the ascending aorta. In the neonatal ductus, SMCs of the media and outer intima were well differentiated and comparable with SMCs of the ascending aorta. Dedifferentiated SMCs, with a low expression of cytoskeletal and contractile proteins and a high expression of EDA and EDB, were found in regions in the inner intima that show features of progression of intimal thickening and in areas of cytolytic necrosis in the media. With a technique using in situ end labeling of DNA fragments, we found extensive apoptosis in the area of cytolytic necrosis and to a lesser extent in these areas of the inner intima. In conclusion, SMCs of the fetal ductus arteriosus have an advanced differentiation of the contractile apparatus compared with the adjacent aorta. Reexpression of fetal characteristics is seen in a number of cells in inner intima and media of the neonatal ductus arteriosus. The finding of apoptosis in these areas suggests that dedifferentiation and apoptosis are associated processes that may play a role in vascular remodeling.
Assuntos
Apoptose , Diferenciação Celular , Canal Arterial/embriologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/embriologia , Aorta/citologia , Aorta/embriologia , Aorta/crescimento & desenvolvimento , Fragmentação do DNA , Canal Arterial/citologia , Canal Arterial/crescimento & desenvolvimento , Feto/irrigação sanguínea , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Hibridização In Situ , Recém-Nascido , Desenvolvimento Muscular , Músculo Liso Vascular/crescimento & desenvolvimentoRESUMO
The origin of myofibroblasts in stromal reaction has been a subject of controversy. To address this question definitively, we developed techniques for purification and characterization of major stromal cell types. We defined a panel of markers that could, in combination, unequivocally distinguish these cell types by immunocytochemistry, iso-electric focusing, immunoblotting, and two-dimensional gel electrophoresis. We then devised an assay to recapitulate in culture, within two weeks of incubation, critical aspects of the microenvironment in vivo including the typical tissue histology and stromal reaction. When confronted with tumor cells in this assay, fibroblasts readily converted into a graded pattern of myogenic differentiation, strongest in the immediate vicinity of tumor cells. Vascular smooth muscle cells (VSMC), in contrast, did not change appreciably and remained coordinately smooth muscle differentiated. Midcapillary pericytes showed only a slight propensity for myogenic differentiation. Analysis of ten primary tumors implicated converted fibroblasts (10/10), vascular smooth muscle cells (4/10), and pericytes (1/10) in the stromal reaction. Tumor cells were shown to specifically denude the venules both in culture and in vivo, explaining the VSMC phenotype in the stroma. The establishment of this assay and clarification of the origin of these cells pave the way for further analysis of the mechanisms of conversion, and of the consequence of such heterogeneity for diagnosis and treatment.
Assuntos
Vasos Sanguíneos/patologia , Neoplasias da Mama/patologia , Biomarcadores/análise , Mama/citologia , Mama/patologia , Neoplasias da Mama/irrigação sanguínea , Separação Celular/métodos , Sulfatos de Condroitina/análise , Proteínas do Citoesqueleto/análise , Epitélio/patologia , Fator VIII/análise , Fibroblastos/patologia , Antígenos HLA-DQ/análise , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Integrinas/análise , Queratinas/análise , Organoides/patologia , Organoides/ultraestrutura , Células Tumorais CultivadasRESUMO
Using five monoclonal antibodies raised against a human uterine smooth muscle extract, we have identified a novel antigen which runs as a closely spaced doublet in SDS-gels. The proteins (60/63 kDa) co-purify, are present in a 1:1 ratio as judged by Coomassie Blue staining, and are immunologically closely related, if not identical. No N-terminal sequence could be obtained from a mixture of the 60/63 kDa proteins, but the sequence of four polypeptides liberated by V8 protease or cyanogen bromide cleavage showed that the proteins are closely related to the glycolytic enzyme phosphoglucomutase type 1. Affinity-purified polyclonal antibodies and three different monoclonal antibodies to the 60/63 kDa proteins cross-reacted with rabbit skeletal muscle phosphoglucomutase type 1, whilst two additional monoclonal antibodies were specific for the 60/63 kDa proteins. Peptide maps of the 60/63 kDa proteins and phosphoglucomutase 1 are markedly different, and the purified proteins have no detectable phosphoglucomutase activity. Staining of cultured smooth muscle cells and fibroblasts with antibodies to 60/63 kDa proteins showed that the antigen is concentrated in focal contacts at the ends of actin bundles and is also associated with actin filaments. About 60% of the cellular 60/63 kDa proteins were found in the detergent-insoluble fraction, suggesting a physical association with the cytoskeleton. The highest levels of protein immunoreactivity were found in muscles. The antigen is concentrated in muscle adherens junctions, including smooth muscle dense plaques, cardiomyocyte intercalated disks, and striated muscle myotendinous junctions. Among epithelial cells, the 63 kDa isoform of the protein was found only in cultured keratinocytes where immunofluorescent staining was localized in cell-to-cell adherens junctions. Expression of the 60/63 kDa proteins in vascular smooth muscle cells is developmentally regulated and correlates with the differentiated contractile phenotype of these cells.
Assuntos
Junções Intercelulares/enzimologia , Músculo Liso Vascular/enzimologia , Músculo Liso/enzimologia , Músculos/enzimologia , Fosfoglucomutase/análise , Adulto , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Aorta/enzimologia , Western Blotting , Células Cultivadas , Criança , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Feminino , Feto , Fibroblastos/enzimologia , Humanos , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fosfoglucomutase/química , Fosfoglucomutase/isolamento & purificação , Gravidez , Coelhos , Homologia de Sequência de Aminoácidos , Útero/enzimologiaRESUMO
The myosin heavy-chain (MHC) composition of developing and adult human aortic smooth muscle (SM) was studied by SDS-polyacrylamide gel electrophoresis, Western blotting and indirect immunofluorescence using a panel of anti-MHC antibodies. On 5% SDS gels, three bands of 204, 200 and 196 kDa apparent molecular mass were identified in fetal, infant and adult stages of development. In the extracts from thoracic aorta (upper level), the 204, and 200-kDa bands (designated as SM-1 and SM-2, respectively) were recognized by SM-G4 and SMMS-1 antibodies, raised against a SM antigen, whereas the 196-kDa band was reactive with nonmuscle (NM)-F6 and NM-G2 antiplatelet MHC antibodies. Western blotting and immunofluorescence tests performed on bovine brain and other human NM tissues using NM-F6 and NM-G2 indicated that antigenic targets of the two antibodies resembled that of so-called IIB and IIA NM myosin found in the bovine system, respectively. In the aortic media, SM-1 was expressed throughout development, while SM-2 was upregulated during late fetal and postnatal development. Similarly, the 196-kDa band showed two distinct patterns of immunoreactivity with the anti-NM-MHC antibodies: with NM-G2, antigenicity was equal at all the developmental stages examined, whereas with NM-F6, it diminished during postnatal development. In the upper level, the cellular distribution of NM-G2 and NM-F6 immunoreactivities was similar in the early fetus but quite distinct at later stages of development. In infant and adult subjects, SM cells (SMC) reactive with NM-F6 accumulated predominantly within the intimal layer as well as in some areas of the underlying media as cell foci, whereas NM-G2 homogeneously stained the two layers. In the aorta near the diaphragm (lower level), both antibodies stained the thickened intima but not the underlying media. These data are consistent with the existence of developmental, stage-specific molecular and cellular transitions during vascular SMC maturation in human aortic media. In addition, these data suggest that IIB-like myosin may be expressed in SMC involved specifically in intimal thickening.
Assuntos
Envelhecimento/metabolismo , Aorta/embriologia , Aorta/enzimologia , Feto/metabolismo , Miosinas/metabolismo , Adulto , Aorta/citologia , Western Blotting , Densitometria , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Humanos , Lactente , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/enzimologia , Miosinas/química , Distribuição TecidualRESUMO
In human long-term marrow cultures connective tissue-forming stromal cells are an essential cellular component of the adherent layer where granulomonocytic progenitors are generated from week 2 onward. We have previously found that most stromal cells in confluent cultures were stained by monoclonal antibodies directed against smooth muscle-specific actin isoforms. The present study was carried out to evaluate the time course of alpha-SM-positive stromal cells and to search for other cytoskeletal proteins specific for smooth muscle cells. It was found that the expression of alpha-SM in stromal cells was time dependent. Most of the adherent spindle-shaped, vimentin-positive stromal cells observed during the first 2 weeks of culture were alpha-SM negative. On the contrary, from week 3 to week 7, most interdigitated stromal cells contained stress fibers whose backbone was made of alpha-SM-positive microfilaments. In addition, in confluent cultures, other proteins specific for smooth muscle were detected: metavinculin, h-caldesmon, smooth muscle myosin heavy chains, and calponin. This study confirms the similarity between stromal cells and smooth muscle cells. Moreover, our results reveal that cells in vivo with the phenotype closest to that of stromal cells are immature fetal smooth muscle cells and subendothelial intimal smooth muscle cells; a cell subset with limited development following birth but extensively recruited in atherosclerotic lesions. Stromal cells very probably derive from mesenchymal cells that differentiate along this distinctive vascular smooth muscle cell pathway. In humans, this differentiation seems crucial for the maintenance of granulomonopoiesis. These in vitro studies were completed by examination of trephine bone marrow biopsies from adults without hematologic abnormalities. These studies revealed the presence of alpha-SM-positive cells at diverse locations: vascular smooth muscle cells in the media of arteries and arterioles, pericytes lining capillaries, myoid cells lining sinuses at the abluminal side of endothelial cells or found within the hematopoietic logettes, and endosteal cells lining bone trabeculae. More or less mature cells of the granulocytic series were in intimate contact with the thin cytoplasmic extensions of myoid cells. Myoid cells may be the in vivo counterpart of stromal cells with the above-described vascular smooth muscle phenotype.
Assuntos
Células da Medula Óssea , Músculo Liso Vascular/citologia , Actinas/metabolismo , Biópsia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Diferenciação Celular , Células Cultivadas , Humanos , Técnicas Imunológicas , Técnicas In Vitro , Proteínas dos Microfilamentos , Miosinas/metabolismo , Fatores de Tempo , Vimentina/metabolismo , Vinculina/metabolismo , CalponinasRESUMO
We have previously described a variant form of the integrin beta 1 subunit (beta 1B)1 characterized by an altered sequence at the cytoplasmic domain. Using polyclonal antibodies to a synthetic peptide corresponding to the unique sequence of the beta 1B, we analyzed the expression of this molecule in human tissues and cultured cells. Western blot analysis showed that the beta 1B is expressed in skin and liver and, in lower amounts, in skeletal and cardiac muscles. The protein was not detectable in brain, kidney, and smooth muscle. In vitro cultured keratinocytes and hepatoma cells are positive, but fibroblasts, endothelial cells, and smooth muscle cells are negative. An astrocytoma cell line derived from immortalized fetal astrocytes was found to express beta 1B. In these cells beta 1B represent integral of 30% of the beta 1 and form heterodimers with alpha 1 and alpha 5 subunits. To investigate the functional properties of beta 1B, the full-length cDNA coding for this molecule was transfected into CHO cells. Stable transfectants were selected and the beta 1B was identified by a mAb that discriminate between the transfected human protein and the endogenous hamster beta 1A. Immunoprecipitation experiments indicated that the beta 1B was exported at the cell surface in association with the endogenous hamster alpha subunits. The alpha 5/beta 1B complex bound to a fibronectin-affinity matrix and was specifically released by RGD-containing peptides. Thus beta 1B and beta 1A are similar as far as the alpha/beta association and fibronectin binding are concerned. The two proteins differ, however, in their subcellular localization. Immunofluorescence studies indicated, in fact, that beta 1B, in contrast to beta 1A, does not localize in focal adhesions. The restricted tissue distribution and the distinct subcellular localization, suggest that beta 1B has unique functional properties.
Assuntos
Citoplasma/metabolismo , Integrinas/fisiologia , Fragmentos de Peptídeos/fisiologia , Sequência de Aminoácidos , Animais , Western Blotting , Células CHO , Células Cultivadas , Cricetinae , Imunofluorescência , Humanos , Integrina beta1 , Integrinas/química , Integrinas/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Testes de Precipitina , Transfecção , Células Tumorais CultivadasRESUMO
The expression of several differentiation markers in normal human mammary gland myoepithelium and in certain stromal fibroblasts ("myofibroblasts") associated with breast carcinomas was studied by immunofluorescence microscopy of frozen sections. Several antibodies to smooth muscle-specific proteins (smooth muscle alpha-actin, smooth muscle myosin heavy chains, calponin, alpha 1-integrin, and high molecular weight caldesmon) and to epithelial-specific proteins (cytokeratins, E-cadherin, and desmoplakin) were used to show that myoepithelial cells concomitantly express epithelial and smooth muscle markers whereas adjacent luminal cells express only epithelial markers. The same antibodies were used to establish that stromal myofibroblasts exhibit smooth muscle phenotypic properties characterized by the expression of all the smooth muscle markers examined except for high molecular weight caldesmon. In addition, both myoepithelium and myofibroblasts show a significant degree of heterogeneity in smooth muscle protein expression. Thus, myoepithelial cells and stromal myofibroblasts are epithelial and mesenchymal cells, respectively, which coordinately express a set of smooth muscle markers while maintaining their specific original features. The dual nature of myoepithelial cells and the phenotypic transition of fibroblasts to myofibroblasts are examples of the plasticity of the differentiated cell phenotype.
Assuntos
Antígenos de Diferenciação/isolamento & purificação , Neoplasias da Mama/química , Mama/química , Proteínas Musculares/isolamento & purificação , Músculo Liso/química , Mama/anatomia & histologia , Neoplasias da Mama/patologia , Epitélio/química , Feminino , Fibroblastos/química , Imunofluorescência , Secções Congeladas , Humanos , Microscopia de FluorescênciaRESUMO
Rabbit aortic smooth muscle cells (SMC) were seeded at moderate or high densities and grown either in the presence of serum or in the serum-substitution formula Monomed. Expression and synthesis of marker proteins caldesmon, calponin, smooth muscle myosin, and vinculin were monitored during SMC cultivation. Contractility was tested by the ability of cultured SMC to deform silicone membranes following ionomycin treatment. The results show that cells of moderate density grown in Monomed, as opposed to those grown in 5% serum, have the smooth muscle isoform of caldesmon 1.6-fold higher, calponin 1.4-fold and smooth muscle myosin 1.4-fold higher on Day 14 of cultivation. Synthesis of these proteins corresponded to their expression in SMC. The metavinculin:vinculin ratio slightly decreased over the first days with a following reestablishment on Day 8. Contraction was observed until Day 13, compared with Day 7 for cells grown in the presence of serum. High seeding density also prevented a decrease in the expression of smooth muscle markers with the exception of smooth muscle caldesmon whose content in the high density SMC culture was not significantly different from that in the moderate density culture. The period of contractility of SMC in the high density culture was also similar to that in the moderate density culture in the presence of serum. We conclude that cultivation of primary SMC in Monomed allows the maintenance of cells in the contractile phenotype more effectively than high initial seeding density.
Assuntos
Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Aorta , Sangue , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Células Cultivadas , Meios de Cultura , Ionomicina/farmacologia , Proteínas dos Microfilamentos , Miosinas/metabolismo , Coelhos , Vinculina/metabolismo , CalponinasRESUMO
The melting of human plasma fibronectin and its proteolytic fragments has been studied by scanning microcalorimetry to reveal co-operative structural domains in the molecule. It has been established that each of the two similar polypeptide chains of fibronectin has at least 12 structural domains, which differ in stability, size and function. Many of the domains in the N-terminal half of the polypeptide chains appear to be composed of two homologous repeat modules that co-operate to form a single co-operative unit. In the intact fibronectin molecule, the C-terminal regions of both chains seem to interact forming a stable co-operative block.
Assuntos
Fibronectinas/sangue , Varredura Diferencial de Calorimetria , Heparina/metabolismo , Humanos , Substâncias Macromoleculares , Modelos Estruturais , Peso Molecular , Fragmentos de Peptídeos , Ligação Proteica , Conformação Proteica , Desnaturação ProteicaRESUMO
Phorbolester (PMA) and forskolin (FSK) cause a dramatic reorganization of microfilaments in cultured cells. We have incubated human blood platelets with PMA and FSK and we investigated the G-/F-actin equilibrium by the DNase I inhibition assay. PMA incubation (0.8 microM, 5 min, 37 degrees C) leads to an increase of filamentous actin (14.4 +/- 4.0%) compared to control platelets. The effect is rapid, dose-dependent and specific, since the biologically inactive derivative phorbol 12,13-didecanoate has no effect. FSK incubation (4 microM, 5 min, 37 degrees C) causes a decrease of F-actin (12.8 +/- 9.0%), the effect is rapid and dose-dependent too. Since 8-bromoadenosine 3':5' cyclic monophosphate also decreases F-actin in human blood platelets, the FSK effect seems to be mediated by cyclic AMP due to affecting the adenylate cyclase.
Assuntos
Actinas/sangue , Colforsina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Células Cultivadas , AMP Cíclico/sangue , Desoxirribonuclease I , Humanos , CinéticaRESUMO
A new protein was found at the site of interaction of cytoskeletal filaments and the plasma membrane in desmosomes of human cardiac muscle intercalated discs. As revealed by the indirect immunofluorescence method, monoclonal antibody XVE2 was able to stain intercalated discs of cardiac muscle and desmosomes of human skin epidermal cells, whereas it did not react with sections from human uterine smooth muscle, vascular tissue and liver. Western immunoblot analysis of extracts of total human heart and uterus demonstrated that a doublet of 65 kDa and 70 kDa polypeptides were the major proteins recognized by the monoclonal antibody XVE2. These 65 kDa and 70 kDa proteins are immunologically distinct from other known intercalated disc proteins such as vinculin, meta-vinculin, filamin, talin, alpha-actinin, desmin and desmoplakins. The distribution of the XVE2 monoclonal antibody antigens raises the possibility that these polypeptides are involved in linking intermediate filaments to the dense plaque of desmosomes of cardiac muscle intercalated discs.