RESUMEN
In this review article we discuss some of the key aspects concerning the development of a polymer-based nanoparticle formulation for intravenous drug delivery. Since numerous preparations fail before and during clinical trials, our aim is to emphasize the main issues that a nanocarrier has to face once injected into the body. These include biocompatibility and toxicity, drug loading and release, nanoparticle storage and stability, biodistribution, selectivity towards the target organs or tissues, internalization in cells and biodegradability. They represent the main checkpoints to define a polymer-based formulation as safe and effective. Indeed, this review is intended to provide guidelines to be followed in the early development of a new nanotherapeutic to hopefully increase the success rate of polymer-based formulations entering clinical trials. The corresponding requirements and characteristics are discussed in the context of some relevant case studies taken from the literature and mainly related to the delivery of lipophilic anticancer therapeutics.
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Antineoplásicos , Portadores de Fármacos , Nanopartículas , Neoplasias/tratamiento farmacológico , Polímeros , Administración Intravenosa , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Portadores de Fármacos/síntesis química , Portadores de Fármacos/química , Portadores de Fármacos/uso terapéutico , Humanos , Nanopartículas/química , Nanopartículas/uso terapéutico , Neoplasias/metabolismo , Neoplasias/patología , Polímeros/síntesis química , Polímeros/química , Polímeros/uso terapéuticoRESUMEN
Ethology has shown that animal groups or colonies can perform complex calculation distributing simple decision-making processes to the group members. For example ant colonies can optimize the trajectories towards the food by performing both a reinforcement (or a cancellation) of the pheromone traces and a switch from one path to another with stronger pheromone. Such ant's processes can be implemented in a photonic hardware to reproduce stigmergic signal processing. We present innovative, completely integrated X-junctions realized using solitonic waveguides which can provide both ant's decision-making processes. The proposed X-junctions can switch from symmetric (50/50) to asymmetric behaviors (80/20) using optical feedbacks, vanishing unused output channels or reinforcing the used ones.
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Hormigas/fisiología , Conducta Animal/fisiología , Feromonas/metabolismo , Animales , Hormigas/metabolismo , Toma de Decisiones , Retroalimentación Fisiológica , Refuerzo en Psicología , Aprendizaje EspacialRESUMEN
One of the major drawbacks that limits the clinical application of nanoparticles is the lack of preliminary investigations related to their biocompatibility, biodegradability and biodistribution. In this work, biodegradable PEGylated polymer nanoparticles (NPs) have been synthesized by using macromonomers based on poly(ε-caprolaconte) oligomers. More in detail, NPs have been produced by adopting a surfactant-free semibatch emulsion polymerization process using PEG chains as a stabilizing agent. The NPs were also labeled with rhodamine B covalently bound to the NPs to quantitatively study their biodistribution in vivo. NPs were investigated in both in vitro and in vivo preclinical systems to study their biodistribution in mice bearing B16/F10 melanoma, as well as their biocompatibility and biodegradability. The NP concentration was evaluated in different tissues at several times after intravenous injection. The disappearance of the NPs from the plasma was biphasic, with distribution and elimination half-lives of 30 min and 15 h, respectively. NPs were retained in tumors and in filter organs for a long time, were still detectable after 7 d and maintained a steady concentration in the tumor for 120 h. 48 h after injection, 70 ± 15% of the inoculated NPs were excreted in the feces. The favorable tumor uptake, fast excretion and absence of cytotoxicity foster the further development of produced NPs as drug delivery carriers.
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Sistemas de Liberación de Medicamentos , Nanopartículas/química , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Tamaño de la Partícula , Polímeros , Rodaminas/química , Rodaminas/farmacocinética , Distribución TisularRESUMEN
Addition reactions of carbon-centered radicals to unsaturated compounds have been studied using quantum chemistry. Following the review by Fischer and Radom (Angew. Chem., Int. Ed. 2001, 40, 1340.), the radicals were grouped in four different families, and the alkenes were selected from among those typical of polymer productions. All of the kinetic constants were calculated using density functional theory and classic transition state theory. Geometries of reactants, products, and transition states were determined at the B3LYP/6-311+G(d,p) level of theory, whereas reaction enthalpies, activation energies, and kinetic constants were estimated using different basis sets. By comparative evaluation of the results obtained with different basis sets, the best computational approach for each kinetic step was identified. As a result of this study, a computational methodology suitable for investigating a large number of kinetic pathways typical of free-radical polymerization processes is proposed.
RESUMEN
In the framework of resorbable devices, an increased demand for a better reliability in drug delivery systems emerged, based on resorbable polymers degradation control. These points influenced researchers to replace the trial-and-error approaches with model-based ones. In this context, we developed a transient 1-dimension model for degradation and drug release of resorbable polymeric drug loaded systems. Such model is thought to be applied to design specific devices based on the expected degradation time and drug delivery rates. It is based on the detailed conservation equations taking into account main physical-chemical parameters involved in polymer degradation and in drug release, all independently estimated. The model leads to a system of partial differential equations solved numerically, which predictions were verified through both literature and our data. Data of different authors, various systems and directly generated data were matched by model predictions. Reliability of parameter estimation procedures was also confirmed by accelerated life tests.
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Sistemas de Liberación de Medicamentos , Preparaciones Farmacéuticas/administración & dosificación , Polímeros/química , Difusión , Composición de Medicamentos , Hidrólisis , Modelos Teóricos , SolubilidadRESUMEN
Questionnaires were distributed to the responsibles and to the dependents of structures with high turnover of people, to have informations about numbers and typology of instruments, habits of dependents and type of cleanings. In these structures, microbiologic quality of air and of informatic instruments' surfaces were evaluated and efficacy of treatment with some products of cleaning and sanification was verified. Air microbiological contamination was comparable to that found in other similar researchs. Fecal contamination indicators were not found on instruments' surfaces, both before, and after treatment; in 8 cases (3%) Staphylococcus aureus was isolated and in 6 cases (2%) species of not pathogenous staphylococci were isolated. At the beginning of the working day, mean values of total aerobic bacterial count at 22 degrees and 37 degrees, were low, settled to zero after treatment and increased progressively during the week. Infective risk for workers can be considered insignificant because of low microbiological contamination. Specific sanification products seem not to be necessary, since common products of cleaning have the same efficacy.
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Microbiología del Aire/normas , Contaminación de Equipos , Lugar de Trabajo , Desinfección , Italia , Encuestas y Cuestionarios , Salud UrbanaRESUMEN
AIM: The aim of this study was to survey, from a microbiological point of view, dental unit water, air and surface quality in public dental offices and in control environments in Milan. METHODS: We studied tap and dental unit water (from fountain, air-water syringe, turbine), at the beginning and at the end of monday and thursday morning activity; air quality with surface air system (SAS) in dental and control offices; handpieces holder, fountain block, arm of the light, dental trolley, inner and outer walls surfaces quality. RESULTS: Water from the dental unit waterline shows average exceeding the law limits 2-3 fold for total bacteriological counts at 37 degrees and 22 degrees, in all examined points, at the beginning of the working day. There is an improvement in the water contamination at the end of the activity. Pseudomonas aeruginosa is frequently found, total and fecal coliforms are absent, while Legionella pneumophila was found only in one control. Air and surfaces quality is quite good, especially in places with ventilation systems in function. CONCLUSION: Dental unit water is the most critical point among those monitored. Water quality has to be improved with specific projects.
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Microbiología del Aire , Bacterias/aislamiento & purificación , Consultorios Odontológicos , Microbiología del AguaRESUMEN
In the presence of FGF-2, cells in suspension expressing FGF receptor-1 will attach to monolayers of cells expressing heparan sulfates. This attachment provides physical evidence for the formation of a trimolecular complex between FGF-2, heparan sulfate, and FGF receptors. We have used this system to determine if receptor isoforms containing or lacking the first of three immunoglobulin-like domains are equally able to form complexes with FGF-2 and heparan sulfates. In the presence of FGF-2, cells expressing either isoform of the receptor were able to attach to monolayers of CHO cells expressing heparan sulfates. No attachment was observed in the absence of FGF-2 or if heparin was included in the incubation medium. Attachment of cells expressing the two receptor isoforms occurred at similar concentrations of FGF-2, and similar concentrations of heparin were required to disrupt the interactions. Thus, there appeared to be little difference between these receptor isoforms in their ability to form trimolecular complexes with FGF-2 and cell-associated heparan sulfates. We also found that, in the presence of FGF-2, cells expressing FGF receptor-1 are able to form complexes with both extracellular matrix and cell-surface heparan sulfates.
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Matriz Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/fisiología , Proteoglicanos de Heparán Sulfato/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Células CHO , Adhesión Celular/fisiología , Cricetinae , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Ratones , Isoformas de Proteínas/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , TransfecciónRESUMEN
A prostatic smooth muscle cell line (PSMC1) was established from the dorsolateral prostate of p53 null mice. The cell line is nontumorigenic when inoculated subcutaneously, under the renal capsule or intraprostatically in syngeneic mice. These cells express alpha-smooth muscle actin (alpha-SMA), indicating their smooth muscle origin, and TGF-beta significantly enhances expression of alpha-SMA. The cells express both androgen receptor (AR) mRNA and protein, and respond mitogenically to physiological concentrations of androgens. PSMC1 cells produce significant amounts of TGF-beta, which stimulates growth by an autocrine mechanism. Dihydrotestosterone (DHT) increases proliferation of PSMC1 cells by promoting TGF-beta secretion. Considering the significant inhibitory effect of TGF-beta on prostatic epithelial cells and its stimulatory effect on the PSMC1 cells, we postulate that TGF-beta produced by prostatic smooth muscle cells may have a paracrine effect on the prostatic epithelium. We also postulate that TGF-beta may be involved in the etiology of benign prostatic hyperplasia (BPH) by stimulating excessive stromal proliferation. Line PSMC1 is the first reported androgen-responsive murine smooth muscle cell line. It will be useful for in vivo and in vitro experiments to study the mechanisms of androgen action on prostatic stroma and for delineating the interactions that occur between prostatic smooth muscle and epithelium that may lead to prostatic diseases such as BPH.
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Andrógenos/fisiología , Comunicación Autocrina/fisiología , Músculo Liso/citología , Músculo Liso/fisiología , Próstata/citología , Próstata/fisiología , Factor de Crecimiento Transformador beta/fisiología , Andrógenos/farmacología , Animales , Línea Celular , Masculino , Ratones , Receptores Androgénicos/fisiologíaRESUMEN
Two prostatic epithelial lines, one of basal origin and one of luminal origin, were established from the dorsolateral prostates of p53 null mice. The cell lines are nontumorigenic when inoculated subcutaneously under the renal capsule or intraprostatically in syngeneic mice. The luminal cell line (PE-L-1) expresses cytokeratins 8 and 18 and the basal cell line (PE-B-1) expresses cytokeratins 5 and 14. The basal cells require serum for growth, whereas the luminal cells grow only in serum-free medium. Both cell lines require the presence of growth factors for optimal growth in culture, with EGF and FGF-2 having the greatest effect on the growth rate. Both lines express androgen receptor (AR) mRNA and protein. Androgen stimulates growth of the basal cell line, indicating that the ARs are functional, whereas growth of the luminal cells is unaffected by androgens. The luminal line is significantly inhibited by exogenous TGF-beta and produces low levels of endogenous TGF-beta. In contrast, the basal cell line produces significant amounts of TGF-beta and its growth is not influenced by this cytokine. Coculture of luminal cells with prostatic smooth muscle cells results in the generation of increased levels of biologically active TGF-beta, indicating a paracrine mechanism of TGF-beta activation that may be involved in the maintenance of normal prostatic function. To our knowledge this is the first report describing both basal and luminal prostatic cell lines from a single inbred animal species and the first indication that prostatic epithelial and stromal cells interact to generate the biologically active form of TGF-beta. These lines will provide an important model for determining basal/luminal interactions in both in vitro and in vivo assays.
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Células Epiteliales/fisiología , Sustancias de Crecimiento/farmacología , Próstata/fisiología , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Bioensayo , División Celular/efectos de los fármacos , Línea Celular , Toxina del Cólera/farmacología , Técnicas de Cocultivo , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Genes p53 , Hidrocortisona/farmacología , Insulina/farmacología , Queratinas/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Próstata/citología , Próstata/efectos de los fármacos , Receptores Androgénicos/análisis , Receptores Androgénicos/genética , Receptores de Factores de Crecimiento Transformadores beta/análisis , Receptores de Factores de Crecimiento Transformadores beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
BACKGROUND: Cathepsin S is a member of the family of cysteine lysosomal proteases. The distribution of cathepsin S is restricted to cells from the mononuclear lineage both in the brain and in the periphery. Also, its protease activity is uniquely stable at neutral pH. MATERIALS AND METHODS: We compared the expression of cathepsin S, B, and L mRNAs in various undifferentiated and differentiated cells of mononuclear origin, and examined the modulation of these mRNAs by inflammatory mediators (lipopolysaccharide and various cytokines). In addition, the effect of these agents on cathepsin S protein levels and protease activity was also determined. Lastly, the ability of cathepsin S to process basement membrane components such as heparan sulfate proteoglycans in vitro and in vivo was assessed. RESULTS: Cathepsin S, B, and L mRNAs are expressed in mature macrophages and microglial cells and not in undifferentiated monocytes. Activators of macrophages negatively regulate all three transcripts. Consistent with this, treatment with these agents leads to a decrease in intracellular cathepsin S protein levels and activity. However, the same treatments result in stimulation of secreted cathepsin S activity. Cathepsin S is capable of degrading heparan sulfate proteoglycans in vitro. Also, when expressed in endothelial cells, cathepsin S autocrinely attenuates the basic fibroblast growth factor (bFGF)-mediated binding of FGF receptor containing cells to endothelial cells, by acting on basement membrane proteoglycans. CONCLUSIONS: Taken together, these data imply that cathepsin S is a regulatable cysteine protease that plays a role in the degradation of extracellular proteins, whose secretion from macrophages and microglia is increased by signals that lead to activation of these cells, and may be important in regulating extracellular matrix interactions. http://link.springer-ny. com/link/service/journals/00020/bibs/5n5p320.html
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Catepsinas/metabolismo , Endopeptidasas , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Microglía/metabolismo , Animales , Northern Blotting , Western Blotting , Catepsina B/genética , Catepsina L , Catepsinas/genética , Catepsinas/farmacología , Adhesión Celular , Línea Celular , Cisteína Endopeptidasas , Inhibidores de Cisteína Proteinasa/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interferón-alfa/metabolismo , Interferón-alfa/farmacología , Interferón gamma/metabolismo , Interferón gamma/farmacología , Interleucina-1/metabolismo , Interleucina-1/farmacología , Leucina/análogos & derivados , Leucina/farmacología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Proteoglicanos/metabolismo , ARN Mensajero , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The roles of heparan sulfate proteoglycans and tyrosine kinase fibroblast growth factor (FGF) receptors in mediating the induction of plasminogen activator (PA) by FGF-2 were investigated using L6 myoblast cells that normally do not express detectable FGF receptors. PA was induced by FGF-2 in a dose-dependent manner in L6 cells expressing transfected FGF receptor-1 but not in nontransfected cells or cells transfected with the vector alone. The PA produced in these cells was characterized as urokinase-type PA (uPA). Thus, expression of a tyrosine kinase FGF receptor was required for induction of uPA. Internalization of FGF through heparan sulfates does not seem to be involved in this response as soluble heparin and suramin at concentrations which inhibited FGF-2 binding to heparan sulfates but not receptors did not affect the induction of uPA by FGF-2. Mutant receptors in which the tyrosine kinase was inactivated were not able to respond to FGF-2. In contrast, mutation of the site of phospholipase Cgamma1 (PLCgamma) binding in the receptor, which causes loss of PLCgamma activation, had no effect on uPA induction by FGF-2. These results suggest that PLCgamma activation is not required for induction of uPA by FGF-2.
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Factor 2 de Crecimiento de Fibroblastos/farmacología , Isoenzimas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Fosfolipasas de Tipo C/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Cloratos/farmacología , Activación Enzimática , Heparina/farmacología , Humanos , Mutagénesis Sitio-Dirigida , Fosfolipasa C gamma , Suramina/farmacologíaRESUMEN
Basic fibroblast growth factor (bFGF) has been identified as an important cytokine for blood cells. To determine whether hematopoietic cells have receptors that recognize bFGF, the ability of human leukemia cell lines to bind 125I-bFGF was investigated. Specific bFGF-binding sites were identified on K562 and HL60 cells, but not on U937 cells. DAMI cells bound low amounts of 125I-bFGF specifically. Binding of 125I-bFGF to K562 cell surfaces was reduced in a dose-dependent manner by unlabeled bFGF or by heparin. Scatchard analysis of binding to K562 cells revealed two classes of binding sites: 1,650 high affinity binding sites per cell with a dissociation constant (kd) of 192 pmol/L, and 36,600 low affinity sites per cell with a kd of 9.3 nmol/L. Chemical crosslinking experiments with K562, HL60, and DAMI cells revealed receptor-growth factor complexes with molecular masses of 140 to 160 kD, similar in size to complexes formed by known receptor species. Binding of 125I-bFGF to K562 cells was sensitive to heparinase treatment but not to chondroitinase treatment, suggesting that heparan sulfate proteoglycans (HSPGs) may be responsible for the low affinity binding sites. To further investigate whether K562 cells make HSPG, the incorporation of 35SO4 into proteoglycans was assessed. Metabolically labeled cell-surface proteoglycans with molecular masses of 180 to 300 kD were identified in K562 cells. These proteoglycans were sensitive to heparinase, demonstrating that K562 cells synthesize bFGF-binding HSPG. Treatment of K562 cells with phorbol-12-myristate-13-acetate (PMA) caused a loss of bFGF-binding capacity. This decreased binding capacity reflected a rapid loss of high affinity receptors. The ability to form bFGF-receptor complexes decreased by 65% to 70% within 1 hour and declined continuously thereafter. The decrease in binding of bFGF was not due to an autocrine downregulation of bFGF receptors, because there was no increase in bFGF after PMA treatment as detected by Western blotting, and suramin, which blocks bFGF binding to receptors, did not prevent the loss of receptors after exposure to PMA. In addition, inhibitors of either protein synthesis or protease activity did not prevent the loss of bFGF receptors in PMA-treated cells. In summary, this work demonstrates that leukemia cell lines have receptors that specifically bind bFGF and supports the hypothesis that bFGF acts directly on certain blood cells to stimulate their proliferation.
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Regulación hacia Abajo/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Leucemia/metabolismo , Proteínas de Neoplasias/metabolismo , Proteoglicanos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Animales , Sitios de Unión , Células CHO/efectos de los fármacos , Células CHO/metabolismo , Cricetinae , Células HL-60/patología , Proteoglicanos de Heparán Sulfato , Heparina/farmacología , Humanos , Leucemia Megacarioblástica Aguda/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Linfoma de Células B Grandes Difuso/patología , Megacariocitos/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Receptores de Factores de Crecimiento de Fibroblastos/clasificación , Células Tumorales CultivadasRESUMEN
To determine whether fibroblast growth factor (FGF) has a role in lens development, we have generated transgenic mice expressing a dominant-negative form of the murine FGF receptor-1 (FGFRDN) in the lens. Using the fibre cell-specific alpha A-crystallin promoter to express the FGFRDN, we have asked whether FGF is required for fibre cell differentiation. The transgenic mice display diminished differentiation of fibre cells as indicated by their reduced elongation. In addition, transgenic lenses have an unusual refractile anomaly that morphological and biochemical data show results from the apoptosis of fibre cells in the central region of the lens. These results show that lens fibre cells are dependent on FGF for their survival and differentiation, and demonstrate that growth factor deprivation in vivo can lead to apoptosis.
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Apoptosis/fisiología , Factores de Crecimiento de Fibroblastos/fisiología , Cristalino/embriología , Animales , Epitelio/patología , Factores de Crecimiento de Fibroblastos/genética , Cristalino/patología , Ratones , Ratones Transgénicos , Fenotipo , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Errores de Refracción/patología , Transducción de SeñalRESUMEN
The myeloid 32D cell line, which grows in suspension and does not express FGF receptors or heparan sulfate proteoglycans, was transfected with the cDNA encoding FGF receptor-1 (32D-flg cells). When co-cultured with glutaraldehyde-fixed Chinese hamster ovary (CHO) cells, the 32D-flg cells remained in suspension in the absence of FGF-2 but attached to the CHO monolayer in the presence of 10 ng/ml FGF-2. In contrast, 32D cells transfected with the vector alone did not attach to the CHO monolayer in the presence of FGF-2. FGF-2-dependent attachment of 32D-flg cells was prevented by inclusion of 10 micrograms/ml heparin in the incubation medium and was diminished when CHO mutants in glycosaminoglycan synthesis or wild-type CHO cells treated with heparinase were used, indicating that the attachment occurred through FGF-2 interactions with heparan sulfates on the CHO cells. Attachment of 32D-flg cells to wild-type CHO cells was half-maximal at 0.4 ng/ml FGF-2 and was also observed with FGF-1 but not FGF-4. 32D-flg cells also attached to living CHO cells in a FGF-2-dependent manner, but attachment was transient at 37 degrees C. Induction of new proteins was not required for FGF-2-dependent attachment, since attachment occurred when the co-cultures were incubated at 4 degrees C and when the 32D-flg cells were preincubated with cycloheximide. FGF-2-dependent attachment of 32D-flg cells was also observed with Balb/C 3T3, NIH 3T3, and bovine capillary endothelial cells. We conclude that attachment is due to FGF-2 binding simultaneously to receptors on the 32D-flg cells and heparan sulfates on the CHO monolayers; thus, the FGF-2 acts as a bridge between receptor-expressing cells and heparan sulfate-bearing cells. In addition, induction of DNA synthesis in 32D-flg cells in response to FGF-2 was potentiated by the CHO-associated heparan sulfates to the same extent as by soluble heparin, indicating that this interaction has functional significance.
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Adhesión Celular/fisiología , Endotelio Vascular/fisiología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Heparina/análogos & derivados , Proteoglicanos/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Células 3T3 , Animales , Células CHO , Capilares , Bovinos , Adhesión Celular/efectos de los fármacos , Comunicación Celular , División Celular , Células Cultivadas , Condroitinasas y Condroitín Liasas/farmacología , Cricetinae , Endotelio Vascular/efectos de los fármacos , Glicosaminoglicanos/farmacología , Heparina/metabolismo , Liasa de Heparina , Heparitina Sulfato/biosíntesis , Cinética , Ratones , Ratones Endogámicos BALB C , Polisacárido Liasas/farmacología , Proteínas Tirosina Quinasas Receptoras/efectos de los fármacos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
The activation of latent transforming growth factor-beta (TGF-beta) by vascular endothelial cells (ECs) is regulated by cellular plasminogen activator (PA)/plasmin, transglutaminase (TGase), and latent TGF-beta levels. Because lipopolysaccharide (LPS) has been reported to reduce EC surface plasmin levels by increasing the production of the inhibitor of PA, PA inhibitor-1 (PAI-1), we have tested whether LPS might suppress latent TGF-beta activation in ECs using two different systems, namely, bovine aortic ECs (BAECs) cocultured with smooth muscle cells (SMCs) and BAECs treated with retinol. BAECs were either cocultured with SMCs after treatment with 15 ng/ml LPS or were treated with 2 microM retinol and/or 10 ng/ml LPS, and the expression of PA, surface plasmin, TGase, and the amounts of active and latent TGF-beta secreted into the culture medium were measured. The downregulation of surface PA/plasmin levels with LPS was accompanied by a profound decline of both TGase and latent TGF-beta expression as well as the suppression of surface activation of latent TGF-beta. The effect was dependent on the concentration of LPS and on treatment time. The formation of TGF-beta did not occur in cells maintained in LPS-contaminated culture medium.
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Endotelio Vascular/metabolismo , Lipopolisacáridos/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Aorta/citología , Aorta/metabolismo , Bovinos , Células Cultivadas , Técnicas Citológicas , Endotelio Vascular/citología , Músculo Liso Vascular/citología , Músculo Liso Vascular/embriología , Activadores Plasminogénicos/metabolismo , Transglutaminasas/metabolismo , Vitamina A/farmacologíaRESUMEN
The regulation of cell surface fibroblast growth factor (FGF) receptors during the differentiation of F9 teratocarcinoma cells was investigated. The capacity of F9 cells to bind 125I-basic FGF (FGF-2) increased upon induction of differentiation with dibutyryl cAMP and retinoic acid. No change in binding capacity was observed in the first 24 h after addition of differentiating agents, but a sixfold increase in binding capacity was observed after 48 h and a fivefold increase after 72 h. Scatchard analysis of the binding data indicated that the increased binding of 125I-FGF-2 was due to an increase in the number of receptors with no change in their affinity. When 125I-FGF-2 was cross-linked to cell surface receptors, an increase in FGF-2-receptor complexes with molecular weights of 140,000-160,000 was also observed in the differentiated F9 cells. Undifferentiated F9 cells are known to secrete FGF-4 and cease expression of this molecule upon differentiation. To determine whether the low level of receptors in undifferentiated cells might be related to their production of FGF ligands, the ability of suramin, a drug that can disrupt FGF-receptor interactions, to modulate receptor number on F9 cells was investigated. Suramin treatment increased the 125I-FGF-2 binding capacity of undifferentiated F9 cells threefold but had little effect on the binding capacity of differentiated cells. In addition, antibodies to FGF-4 increased the 125I-FGF-2 binding capacity of undifferentiated F9 cells by 58%. These results suggest that undifferentiated F9 cells might be responding in an autocrine manner to their own FGF ligands resulting in downregulation of cell surface FGF receptors. The increased number of receptors observed in differentiated cells may partly result from the decreased production of FGF ligands by these cells.
Asunto(s)
Regulación hacia Abajo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Teratocarcinoma/metabolismo , Animales , Bucladesina/farmacología , Diferenciación Celular , Reactivos de Enlaces Cruzados/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Ratones , Peso Molecular , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/química , Suramina/farmacología , Teratocarcinoma/patología , Factores de Tiempo , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
Multiple forms of FGF-2 have been shown to exist in many cell types. These different species of molecular masses of 18, 21.5, 22, and 24 kDa are all translated via the use of alternate initiation codons. The three forms of HMW FGF-2 initiate at CUGs codons, whereas the 18 kDa form initiates at an AUG codon. The entire 18 kDa sequence is contained within the larger forms of HMW FGF-2 as the AUG codon is 3' to the CUG codons. Although the 18 kDa form FGF-2 is localized primarily in the cytosol, a significant fraction of the HMW FGF-2 has a nuclear location. The nuclear localization of HMW FGF-2 is determined by amino acid residues in the amino-terminal extended sequence. The residues required for nuclear localization appear to be RG repeats that are found at multiple sites within the amino-terminal extension of HMW FGF-2. The nuclear localization of HMW FGF-2 suggested that these species may have unique properties. By selecting permanent transfectants of 3T3 cells expressing HMW, 18 kDa FGF-2, or all forms of FGF-2, we have found that HMW FGF-2 can endow cells with a phenotype different from that of cells expressing 18 kDa FGF-2. These cells are transformed by what appears to be the intracellular action of HMW FGF-2. The interaction of FGF-2 with heparin has also been examined.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Núcleo Celular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/fisiología , Heparina/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Línea Celular , Codón , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Ratones , Peso Molecular , Mutagénesis Sitio-Dirigida , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
The role of heparin or heparan sulfates in the interaction of basic fibroblast growth factor (bFGF) with its high affinity receptor were investigated using purified extracellular ligand-binding region of FGF receptor-1 (FGFR-1) and intact receptors expressed in a myeloid cell line (32D) that does not express detectable levels of heparan sulfate proteoglycans or in Chinese hamster ovary (CHO) cell mutants defective in heparan sulfate synthesis. The purified extracellular domain of FGFR-1 formed complexes with 125I-bFGF both in the presence or absence of heparin. Intact FGFR-1 expressed in 32D cells also bound the same amount of 125I-bFGF in the presence or absence of heparin when saturating concentrations of bFGF were used. Varying the concentration of 125I-bFGF showed that heparin increased the amount of 125I-bFGF bound at low bFGF concentrations and increased the affinity of bFGF for its receptor by about 3-fold. To eliminate the possibility of alteration of bFGF properties through the chemical modification reactions, bFGF was labeled biosynthetically. The binding of biosynthetically labeled bFGF to FGFR-1 also did not require heparin. When FGFR-1 or FGFR-2 were expressed in mutant CHO cells deficient in heparan sulfate synthesis, the cells also bound 125I-bFGF in the absence of heparin, and the addition of heparin increased the affinity of bFGF for its receptors 2-3-fold. Thus, heparin or heparan sulfate is not required for the binding of bFGF to its receptors but increases the binding affinity to a moderate degree. Finally, the requirement for heparin in signal transduction through the receptor was investigated. Expression of c-fos mRNA was induced by bFGF in 32D cells expressing FGFR-1 to the same extent in the presence or absence of heparin.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparina/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Condroitinasas y Condroitín Liasas/farmacología , Expresión Génica/efectos de los fármacos , Genes fos , Liasa de Heparina , Humanos , Ratones , Polisacárido Liasas/farmacología , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , TransfecciónRESUMEN
The fibroblast growth factor receptor 1 (flg) contains eight acidic amino acids between the first and second immunoglobulin domain. This report examines the role of the acidic domain in the interaction of the flg receptor with its ligands. We observed a marked inhibition of binding of bFGF to the receptor when the acidic domain was completely deleted, but mutants with two and four amino acids deleted (flg delta A2 and flg delta A4, respectively) still bound the ligand. After addition of a bifunctional cross-linking reagent, cross-linked complexes (between bFGF and receptor) with the expected size were observed in cells expressing mutants lacking two or four acidic residues, but not in cells expressing mutants lacking six or eight acidic residues. Immunoprecipitation with anti-flg antibody followed by electrophoresis produced a band of 90 Kd in tunicamycin-treated cells expressing the mutant as well as the wild-type receptors, indicating that the inhibition of binding was not due to defective expression of the protein. The ability of flg delta A8 to mediate a mitogenic response to FGFs was also greatly reduced when this mutated receptor was expressed in receptor-negative cells. The effect of replacing the acidic amino acids with lysine residues was also studied. Binding of bFGF to cells transfected with a plasmid encoding a mutated protein with four amino acid substitutions was totally inhibited, but an eight amino acid substitution did not alter ligand binding to the receptor. In this case the mutation with four amino acids substitution caused a drastic impairment of protein expression. Thus the acidic domain of the FGFR-1 plays an essential role in receptor function, either because it is important for a stable protein configuration or for ligand-receptor interaction.