RESUMO
IGF-I and IGF-II are single-chain polypeptide growth factors that regulate pleiotropic cellular responses. We have characterized the effect of recombinant IGF proteins, as well as third-generation adenoviral vectors encoding either IGF-I or IGF-II genes, on cardiomyocyte apoptosis and on angiogenesis. We found that endothelial cells cultured in the presence of the extracellular protein laminin exhibit a robust response to IGF-I and -II proteins via enhanced cell migration and angiogenic outgrowth. Furthermore, IGF vectors greatly enhanced neovascularization in an in vivo Matrigel model. Transduction of cardiomyocytes with the IGF adenoviral vectors resulted in a dose- and time-dependent increase in the expression of IGF-I or IGF-II protein. This correlated with abrogation of apoptosis induced by ischemia-reoxygenation, ceramide, or heat shock with optimal inhibition of approximately 80%. We conclude that gene transfer of IGF-I and IGF-II is a plausible strategy for the local delivery of IGFs to treat ischemic heart disease and heart failure by stimulating angiogenesis and protecting cardiomyocytes from cell death.
Assuntos
Apoptose , Vetores Genéticos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like I/genética , Miocárdio/citologia , Neovascularização Fisiológica , Adenoviridae/genética , Animais , Aorta , Adesão Celular , Quimiotaxia , Endotélio Vascular/citologia , Fator 2 de Crescimento de Fibroblastos , Expressão Gênica , Terapia Genética , Coração/embriologia , Cardiopatias/terapia , Humanos , Laminina/metabolismo , Isquemia Miocárdica/terapia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Veias UmbilicaisRESUMO
Adenoviral vector-based gene therapy is a promising approach for the treatment of restenosis postangioplasty. However, a high concentration of adenoviral vector can cause cellular activation, damage, and an enhanced immune response. One approach to solving this problem is to increase gene transfer efficiency by directing adenoviral vector entry via an alternate receptor system. We have constructed an adenoviral vector, Av9LacZ, that encodes the beta-galactosidase gene and contains a chimeric fiber protein that redirects viral vector binding to the Ad3 adenoviral receptor on the host cell. We examined the ability of Av9LacZ to transduce primary human smooth muscle cells (SMC) and found that it showed a 10- to 15-fold higher transduction efficiency when compared to the prototypic adenoviral vector currently used for preclinical and clinical studies. While both vectors were able to transduce rabbit, pig and monkey SMCs, the genetically modified vector transduced human SMC with much higher efficiency. SMC obtained from the aorta, coronary, renal, popliteal and pulmonary arteries were all efficiently transduced by Av9LacZ. Consistent with the data obtained from cultured cells, Av9LacZ also transduced fresh human arterial tissues considerably more efficiently than Av1LacZ. We conclude that the large discrepancy between transduction of animal and human cells by conventional vectors supports a cautious extrapolation of the results of in vivo animal studies to man. Furthermore, the genetically modified AV9 vector may deliver better efficacy and studies in large animal models with this vector could be more predictive of therapeutic efficacy in the treatment of human restenosis.
Assuntos
Adenoviridae/genética , Proteínas do Capsídeo , Capsídeo/genética , Vetores Genéticos , Músculo Liso Vascular/metabolismo , beta-Galactosidase/genética , Animais , Antígenos Virais/genética , Aorta , Células Cultivadas , Humanos , Macaca fascicularis , Técnicas de Cultura de Órgãos , Coelhos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , SuínosRESUMO
Gene therapy using recombinant adenoviral vectors represents a promising therapeutic tool to prevent vein graft stenosis, the main complication of coronary artery bypass grafting. However, the low transduction efficiency of vascular smooth muscle cells and endothelial cells (EC) is a potential limitation, presumably due to the low levels of functional adenovirus receptor (coxsackie:adenovirus receptor; CAR). Designing vectors specifically targeted to alpha(v) integrins is a strategy that might overcome the poor expression of CAR in vascular smooth muscle cells and EC. RGD, a receptor-binding motif that can interact with alpha(v) integrins, was inserted into the HI loop and at the C-terminus of the adenoviral fiber protein in two separate adenovirus vectors encoding a beta-galactosidase reporter gene. Av1nBgCRGD (C-terminus) and Av1nBgHIRGD (HI loop) were evaluated in EC in culture and in jugular vein organ culture. Transduction of primary rat and rabbit EC with Av1nBgHIRGD was significantly more efficient when compared to Av1nBgCRGD or Av1nBg. Transduction of mouse, rat and rabbit jugular veins in organ culture using Av1nBg showed that adenovirus-mediated gene expression was greatest in rabbit jugular veins compared to rat and mouse veins. Av1nBgHIRGD augmented gene expression approximately four-fold in rabbit jugular veins when compared to Av1nBg. Histochemical analysis showed that numerous EC but few smooth muscle cells were transduced at all vector concentrations. A substantial number of adventitial fibroblasts were transduced only at the highest vector concentrations of Av1nBgHIRGD. These findings demonstrate that integrin-targeted vectors allow for enhanced gene delivery to veins and strengthen the viability of adenoviral-mediated gene transfer of therapeutic transgenes to human veins prior to vein grafting.
Assuntos
Adenoviridae/genética , Antígenos CD/metabolismo , Proteínas do Capsídeo , Capsídeo/genética , Transferência Genética Horizontal , Veias Jugulares , Receptores Imunológicos/genética , Receptores de Peptídeos/genética , Sequência de Aminoácidos , Animais , Capsídeo/química , Endotélio Vascular/metabolismo , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Histocitoquímica , Integrina alfaV , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Técnicas de Cultura de Órgãos , Coelhos , Ratos , Ratos Sprague-Dawley , Receptores Imunológicos/química , Receptores de Peptídeos/química , Proteínas Recombinantes de Fusão , Especificidade da Espécie , TransfecçãoRESUMO
Endostatin is a cleavage product of collagen XVIII that inhibits tumor angiogenesis and growth. Interferon alpha2a blocks tumor angiogenesis and causes regression of hemangiomas, but has no effect on choroidal neovascularization (CNV). Therefore, inhibitors of tumor angiogenesis do not necessarily inhibit ocular neovascularization. In this study, we used an intravenous injection of adenoviral vectors containing a sig-mEndo transgene consisting of murine immunoglobulin kappa-chain leader sequence coupled to sequence coding for murine endostatin to investigate the effect of high serum levels of endostatin on CNV in mice. Mice injected with a construct in which sig-mEndo expression was driven by the Rous sarcoma virus promoter had moderately high serum levels of endostatin and significantly smaller CNV lesions at sites of laser-induced rupture of Bruch's membrane than mice injected with null vector. Mice injected with a construct in which sig-mEndo was driven by the simian cytomegalovirus promoter had approximately 10-fold higher endostatin serum levels and had nearly complete prevention of CNV. There was a strong inverse correlation between endostatin serum level and area of CNV. This study provides proof of principle that gene therapy to increase levels of endostatin can prevent the development of CNV and may provide a new treatment for the leading cause of severe loss of vision in patients with age-related macular degeneration.
Assuntos
Inibidores da Angiogênese/genética , Corioide/irrigação sanguínea , Colágeno/genética , Terapia Genética/métodos , Neovascularização Patológica/prevenção & controle , Fragmentos de Peptídeos/genética , Adenoviridae/genética , Inibidores da Angiogênese/sangue , Animais , Colágeno/sangue , Colágeno Tipo XVIII , Endostatinas , Expressão Gênica , Genes Reporter/genética , Vetores Genéticos , Injeções Intravenosas , Fígado/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/patologia , Fenômenos Fisiológicos Oculares , Fragmentos de Peptídeos/sangue , Recombinação GenéticaRESUMO
Restenosis from neointimal proliferation is a frequent complication of intracoronary stenting and catheter-based revascularization procedures. Currently, there is no known therapeutic strategy that has been sufficiently effective to warrant its widespread use. In the present study, the anti-proliferative properties of a matrix (collagen)-targeted retroviral vector bearing a mutant cyclin G1 (DNT 41-249) construct was evaluated in vitro and in vivo. In controlled one-month efficacy studies, the intraluminal instillation of the mutant cyclin G1 vector significantly inhibited neointima lesion formation in balloon-injured rat arteries without neointimal growth, associated necrosis or intense inflammatory reaction. Taken together, these data extend the potential utility of the matrix-targeted mutant cyclin G1 retroviral vector for management of vascular restenosis.
Assuntos
Lesões das Artérias Carótidas/prevenção & controle , Ciclinas/administração & dosagem , Túnica Íntima/patologia , Células 3T3 , Sequência de Aminoácidos , Angioplastia com Balão/efeitos adversos , Animais , Artérias Carótidas/química , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/etiologia , Lesões das Artérias Carótidas/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Linhagem Celular , Ciclina G , Ciclina G1 , Ciclinas/análise , Ciclinas/genética , Ciclinas/uso terapêutico , DNA Antissenso/genética , DNA Recombinante/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Humanos , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Mutação , Ratos , Retroviridae/genética , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Resultado do Tratamento , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/metabolismoRESUMO
A gene therapy approach towards the modulation of neovascularization provides important advantages that could be crucial for the success of therapies that target blood vessels. These advantages include sustained local expression and the ability to supply multiple pro- or anti-angiogenic factors. There is potential near-term success in the application of this approach for the treatment of ischemic vascular diseases. Although there is convincing proof of concept in animal models that an anti-angiogenesis gene therapy approach can be used to treat cancer, this is a highly competitive field with small molecules, recombinant proteins and monoclonal antibodies already in clinical trials. The scientific rationale for the use of gene therapy is sound, but realization of its full potential for the treatment of a broad array of diseases will require several challenging technical hurdles to be overcome and safety concerns to be alleviated.
RESUMO
Thrombospondin-1 (TSP1) is a secreted trimeric glycoprotein of 450 kDa with demonstrated effects on cell growth, adhesion and migration. Its complex biological activity is attributed to its ability to bind to cell-surface receptors, growth factors and extracellular-matrix proteins. In this study, we used a (125)I solid-phase binding assay to demonstrate that TSP1 binds specifically to proteins containing polyhistidine stretches. Based on studies with three different six-histidine-containing recombinant proteins, we derived an average dissociation constant of 5 nM. The binding of (125)I-labelled TSP1 to these proteins was inhibited by peptides containing histidine residues, with the degree of competition being a function of the number of histidines within the peptide. Binding was not inhibited by excess histidine or imidazole, indicating that the imidazole ring is not sufficient for recognition by TSP1. Heparin was a potent inhibitor of binding with a K(i) of 50 nM, suggesting that the heparin-binding domain of TSP1 may be involved in this interaction. This was confirmed by the ability of a recombinant heparin-binding domain of TSP1 to directly compete for TSP1 binding to polyhistidine-containing proteins. Affinity chromatography with a polyhistidine-containing peptide immobilized on agarose revealed that TSP1 in platelet releasates is the major polypeptide retained on the six-histidine-peptide column. We conclude that TSP1 contains a high-affinity binding site for polyhistidine and this is likely to be the molecular basis for the observed binding of TSP1 to histidine-rich glycoprotein. The possibility that other polyhistidine-containing proteins also interact with TSP1 warrants further study.
Assuntos
Histidina , Peptídeos/metabolismo , Trombospondina 1/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Plaquetas/química , Plaquetas/metabolismo , Western Blotting , Cálcio/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Cromatografia de Afinidade , Dissulfetos/metabolismo , Heparina/metabolismo , Humanos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Peptídeos/síntese química , Peptídeos/química , Ativação Plaquetária , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Termodinâmica , Trombospondina 1/química , Trombospondina 1/isolamento & purificaçãoRESUMO
The glycosaminoglycan hyaluronan (HA) and HA-binding proteins (HABPs) serve important structural and regulatory functions during development and in maintaining adult tissue homeostasis. Here we have identified and partially characterized the sequence and expression pattern of three putative novel HABPs. DNA sequence analysis revealed that two of the novel HABPs, WF-HABP and BM-HABP, form a unique HA-binding subfamily, whereas the third protein, OE-HABP, is more closely related to the LINK subfamily of HABPs. Northern blotting experiments revealed that the expression of BM-HABP was highly restricted, with substantial expression detected only in human fetal liver. In contrast, WF-HABP and OE-HABP mRNAs were detected in a number of tissues, with particularly prominent expression in highly vascularized tissues such as the heart, placenta, and lung. Additional studies showed that OE-HABP was expressed by cultured human endothelial cells, smooth muscle cells, and differentiated monocytes. However, only endothelial cells expressed WF-HABP mRNA, and its expression was regulated by growth state, being most prominent in quiescent endothelial cells. We further characterized the expression of WF-HABP in vivo and found that its expression colocalized with CD31-positive cells and was prominently expressed in microvessels in the human aorta and in atherectomy samples. Our data suggest that WF-HABP is an endothelial cell-specific HA receptor and that it may serve a unique function in these cells. The WF-HABP gene was localized to chromosome 3p21.31 and the OE-HABP gene to 15q25.2-25.3.
Assuntos
Cromossomos Humanos Par 15 , Cromossomos Humanos Par 3 , DNA Complementar/análise , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Adulto , Sequência de Aminoácidos , Mapeamento Cromossômico , DNA Complementar/genética , Humanos , Receptores de Hialuronatos/metabolismo , Dados de Sequência Molecular , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
Hyaluronan (HA) and HA-binding proteins have been implicated in a diverse array of biological processes, including development, tissue repair, and tumor invasion. However, the role of HA and HA-binding proteins in atherosclerosis and restenosis is poorly understood. PS4 (TSG-6) is a HA-binding protein expressed by cultured vascular smooth muscle cells (SMCs) in response to serum and growth factor stimulation. To delineate a possible role for TSG-6 in vascular disease progression, we have characterized its expression in cultured SMCs and in a rat vascular injury model, and we have studied the effect of constitutive overexpression of TSG-6 on SMC behavior. We found that interleukin-1 (IL-1) but not tumor necrosis factor or interleukin-6 was able to stimulate TSG-6 expression in SMCs. The IL-1 pathway could be distinguished from the growth factor pathway by its insensitivity to protein synthesis inhibitors. Furthermore, epidermal growth factor, fibroblast growth factor-1, and transforming growth factor-beta1 were all capable of augmenting maximum IL-1-induced expression of TSG-6. To gain further insight into the function of TSG-6 in SMCs, we examined the effect of constitutive overexpression of TSG-6 on these cells. We found that TSG-6-overexpressing cells grew >50% faster than control cells. Furthermore, this growth advantage became more evident in the absence of serum growth factors, with an average increase in cell number of 118% over control cells after 6 days. Consistent with these in vitro data, we observed intense immunostaining for TSG-6 in proliferating SMCs in the rat neointima after injury, whereas only an occasional cell was positive for TSG-6 in the medial layer and in nonballooned arteries. We conclude that the expression of TSG-6 is tightly controlled by growth factors and cytokines via two distinct pathways in SMCs and that overexpression of TSG-6 confers a growth advantage to these cells.
Assuntos
Citocinas/farmacologia , Substâncias de Crescimento/farmacologia , Ácido Hialurônico/metabolismo , Músculo Liso Vascular/lesões , Músculo Liso Vascular/metabolismo , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Fator 1 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica/efeitos dos fármacos , Interleucina-1/farmacologia , Músculo Liso Vascular/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Ratos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Thrombospondin-1 (TSP-1) is a large modular trimeric protein that has been proposed to play a diverse role in biological processes. Newly synthesized TSP-1 either is incorporated into the matrix or binds to the cell surface where it is rapidly internalized and degraded. TSP-1 catabolism is mediated by the low density lipoprotein receptor-related protein (LRP), a large endocytic receptor that is a member of the low density lipoprotein receptor family. Using adenovirus-mediated gene transfer experiments, we demonstrate that the very low density lipoprotein receptor can also bind and internalize TSP-1. An objective of the current investigation was to identify the portion of TSP-1 that binds to these endocytic receptors. The current studies found that the amino-terminal heparin binding domain (HBD, residues 1-214) of mouse TSP-1, when prepared as a fusion protein with glutathione S-transferase (GST), bound to purified LRP with an apparent KD ranging from 10 to 25 nM. Recombinant HBD (rHBD) purified following proteolytic cleavage of GST-HBD, also bound to purified LRP, but with an apparent KD of 830 nM. The difference in affinity was attributed to the fact that GST-HBD exists in solution as a dimer, whereas rHBD is a monomer. Like TSP-1, 125I-labeled GST-HBD or 125I-labeled rHBD were internalized and degraded by wild type fibroblasts that express LRP, but not by fibroblasts that are genetically deficient in LRP. The catabolism of both 125I-labeled GST-HBD and rHBD in wild type fibroblast was blocked by the 39-kDa receptor-associated protein, an inhibitor of LRP function. GST-HBD and rHBD both completely blocked catabolism of 125I-labeled TSP-1 in a dose-dependent manner, as did antibodies prepared against the HBD. Taken together, these data provide compelling evidence that the amino-terminal domain of TSP-1 binds to LRP and thus the recognition determinants on TSP-1 for both LRP and for cell surface proteoglycans reside within the same TSP-1 domain. Further, high affinity binding of TSP-1 to LRP likely results from the trimeric structure of TSP-1.
Assuntos
Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Endocitose , Heparina/metabolismo , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Conformação Proteica , Receptores de LDL/metabolismo , Relação Estrutura-Atividade , TrombospondinasRESUMO
Sodium butyrate is a small, naturally occurring molecule with demonstrated activity on cell growth and differentiation. However, its effect on smooth muscle cells had not been examined. We have found that sodium butyrate and its more stable in vivo analogue tributyrin are potent DNA synthesis and cell proliferation inhibitors. The inhibitory activity of sodium butyrate was not mediated by an elevation of endogenous cAMP levels, a known pathway involved in SMC growth-arrest and maintenance of the contractile phenotype. Consistent with the concept that its activity is mediated by a cAMP-independent pathway, butyrate was able to augment the maximum DNA synthesis inhibitory effect of various agents that elevated intracellular cAMP levels. Additionally, butyrate present for just the initial 8 h or present as late as 16 h after serum addition was able to inhibit DNA synthesis. By contrast, the cAMP analogue, 8Br-cAMP had to be present throughout the entire G0 to S phase of the cell cycle to effectively inhibit DNA synthesis. These results indicated that sodium butyrate inhibited SMC growth through a cAMP-independent mechanism. We also found that sodium butyrate was unable to abrogate the expression of the serum-inducible genes c-fos, c-myc, Ki-Ras and PS4, but was able to directly stimulate the expression of PS4 and thrombospondin. These results indicate that a number of important early G1 events initiated by serum growth factors are unaltered by sodium butyrate and that this compound is able to directly stimulate the expression of certain genes normally associated with SMC proliferation.
Assuntos
Butiratos/farmacologia , Regulação da Expressão Gênica , Músculo Liso Vascular/efeitos dos fármacos , Monofosfato de Adenosina/metabolismo , Animais , Aorta , Ácido Butírico , Ciclo Celular , Diferenciação Celular/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , DNA/antagonistas & inibidores , DNA/biossíntese , Genes fos , Genes myc , Genes ras , Glicoproteínas de Membrana/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Coelhos , Trombospondinas , Triglicerídeos/metabolismo , Triglicerídeos/farmacologiaRESUMO
H19 is a developmentally regulated gene with putative tumor suppressor activity, and loss of H19 expression may be involved in Wilms' tumorigenesis. In this report, we have performed in situ hybridization analysis of H19 expression during normal rabbit development and in human atherosclerotic plaques. We have also used cultured smooth muscle cells to identify H19 regulatory factors. Our data indicate that H19 expression in the developing skeletal and smooth muscles correlated with specific differentiation events in these tissues. Expression of H19 in the skeletal muscle correlated with nonproliferative, actin-positive muscle cells. In the prenatal blood vessel, H19 expression was both temporally and spatially regulated with initial loss of expression in the inner smooth muscle layers adjacent to the lumen. We also identified H19-positive cells within the adult atherosclerotic lesion and we suggest that these cells may recapitulate earlier developmental events. These results, along with the identification of the insulin family of growth factors as potent regulatory molecules for H19 expression, provide additional clues toward understanding the physiological regulation and function of H19.
Assuntos
Arteriosclerose/metabolismo , Regulação da Expressão Gênica , Proteínas Musculares/análise , Músculo Liso Vascular/química , RNA não Traduzido , Somatomedinas/fisiologia , Animais , Sequência de Bases , Biomarcadores , Diferenciação Celular , Dados de Sequência Molecular , Proteínas Musculares/genética , Músculo Esquelético/química , Músculo Esquelético/citologia , RNA Longo não Codificante , CoelhosRESUMO
Apoptosis is a physiological cell death process important for normal development and involved in many pathological conditions. In atherosclerosis, pathological accumulation of cells in the intima has been attributed to the migration and proliferation of smooth muscle cells, macrophages, and lymphocytes. In this report, we explored the possibility that apoptosis may also contribute to the pathogenesis of this disease. We examined 35 human atherosclerotic lesion samples and identified a substantial number of cells undergoing apoptosis in 25 of the samples. Furthermore, in a rat vascular injury model, apoptotic cells were specifically identified in the neointima. The presence of apoptotic cells was demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, nuclear staining with propidium iodide, and electron microscopy. Immunostaining with cell-type-specific markers and subsequent terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling analysis on the same sample revealed that the majority of the apoptotic cells were modulated smooth muscle cells as well as macrophages. These results indicate that apoptosis occurs in cells of the injured blood vessel as well as the advanced atherosclerotic lesion and that physiological cell death may have an important role in determining the course of atherogenesis.
Assuntos
Apoptose , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Humanos , Macrófagos/patologia , Microscopia Eletrônica , Pessoa de Meia-Idade , Músculo Liso Vascular/patologia , RatosRESUMO
We are interested in understanding the molecular events associated with the growth-arrest of vascular SMCs. We constructed a subtracted cDNA library enriched in nucleotide sequences associated with quiescent SMCs. This library was screened with similarly subtracted 32P-labeled cDNAs to identify growth-arrest associated cDNA clones. Characterization of 19 of these cDNA clones revealed that 9 hybridized to mRNAs that exhibited a 2-3-fold increase in growth-arrested SMCs. In addition, two other cDNAs hybridized to a 5 Kb mRNA that was elevated approximately 10-fold in high density growth-arrested SMCs. Genomic Southern blot hybridization and DNA sequencing analysis indicated that these cDNAs encoded the same gene (LG7) and that this gene may be a member of a multigene family or that it may contain a sequence shared by other unrelated genes. Augmented expression of LG7 was associated with both high cell density and serum deprivation induced growth-arrest. LG7 mRNA expression was down-regulated when SMCs were incubated with FBS or with reagents that arrest cells in early S-phase. Additional analysis with cell cycle specific inhibitors indicated that LG7 mRNA levels were also low when cells were blocked at the G2 phase of the cell cycle but blockage at mitosis resulted in an elevated level of LG7 mRNA. We further demonstrated that the expression of LG7 was dependent on the presence of a relatively labile protein since protein synthesis inhibitors specifically blocked the expression of this mRNA but not the mRNA expression of alpha 1(III) collagen or ferritin H-chain. Finally, we demonstrated that Bt2cAMP was able to induce mRNA expression of LG7 within 2 h, suggesting that this gene may be directly regulated via the cyclic-AMP-dependent protein kinase pathway.
Assuntos
Ciclo Celular/genética , DNA Complementar/isolamento & purificação , Regulação da Expressão Gênica , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Sequência de Bases , Northern Blotting , Southern Blotting , Bucladesina/farmacologia , Divisão Celular/genética , Células Cultivadas , Colágeno/biossíntese , Meios de Cultura Livres de Soro , DNA Complementar/metabolismo , Ferritinas/biossíntese , Fase G2 , Regulação da Expressão Gênica/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , RNA Mensageiro/biossíntese , Coelhos , Fase SRESUMO
Intimal smooth muscle cell accumulation is regarded as an important component of atherosclerotic plaque formation, angioplasty-induced restenosis, and vascular graft occlusion. Vascular smooth muscle cells can both express and respond to acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF); therefore, under certain conditions these polypeptides may regulate smooth muscle cell growth in an autocrine manner. Previous studies using smooth muscle cells cultured in vitro have identified factors that can enhance aFGF and bFGF gene expression. In this study, we assayed fibroblast growth factor gene expression in a spontaneously immortalized rabbit smooth muscle cell line. In contrast to "normal" rabbit smooth muscle cells, these immortalized cells acquire an altered morphology and enhanced proliferative rate during; cell passaging in vitro. Both "normal" and immortalized rabbit smooth muscle cells express bFGF but not aFGF transcripts. RNA gel blot hybridization, reverse transcription/polymerase chain reaction amplification, and Western blotting techniques demonstrate that bFGF expression in the immortalized smooth muscle cell line increases as a function of passage level. This continuous cell line should prove valuable for studying both the regulation of bFGF synthesis and the control of vascular smooth muscle cell proliferation.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Músculo Liso Vascular/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Fator 2 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Humanos , Immunoblotting , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , CoelhosRESUMO
We have used subtraction cloning to isolate a cDNA (PS4) that identified a serum-inducible mRNA of 1.9 kilobases in rabbit vascular smooth muscle cells. DNA sequence analysis revealed one major open reading frame encoding a 9,442 M(r) protein. Comparison of the DNA as well as the putative protein sequence with various data bases revealed no homology with other sequences. In vitro translation of synthesized PS4 mRNA generated a major polypeptide of 12 kDa. Serum stimulation of quiescent smooth muscle cells in culture induced a rapid increase in the level of PS4 mRNA. Expression of this message was detected by 1 h, peaked at approximately 4 h, and became undetectable by 12 h. The induction of PS4 by serum was completely blocked by cycloheximide, indicating its expression required prior protein synthesis. Epidermal growth factor, acidic fibroblast growth factor, and transforming growth factor-beta 1 also induced a strong increase in PS4 expression. By contrast, platelet-derived growth factor-BB was only able to mildly stimulate the level of PS4 mRNA and insulin-like growth factor-I was unable to enhance PS4 expression. There was a high level of PS4 mRNA in rabbit fetal muscle, esophagus, kidney, and lung, a low level in fetal aorta and heart, and an undetectable level in fetal liver, brain, as well as, in the placenta. The expression of PS4 in the corresponding adult tissues was low or undetectable. Our analysis indicate that PS4 expression is developmentally regulated and tightly controlled by growth factors, suggesting this novel gene has a role in cell growth and differentiation.
Assuntos
DNA/genética , Regulação da Expressão Gênica , Substâncias de Crescimento/farmacologia , Proteínas Musculares/genética , Músculo Liso Vascular/fisiologia , RNA Mensageiro/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Meios de Cultura , Feto , Regulação da Expressão Gênica/efeitos dos fármacos , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Peso Molecular , Músculo Liso Vascular/efeitos dos fármacos , Fases de Leitura Aberta , Especificidade de Órgãos , Poli A/genética , Poli A/isolamento & purificação , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Coelhos , Proteínas Recombinantes/farmacologia , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Repeated cycles of allylamine-induced aortic injury in vivo modulate the proliferative potential of smooth muscle cells (SMCs) during serial propagation in vitro. This modulation may be partly mediated by disturbances in polyphosphoinositide metabolism which afford allylamine-treated cells a growth advantage over control cells [Cox, Murphy and Ramos (1990) Exp. Mol. Pathol. 53, 52-63]. The present studies were conducted to further evaluate the mechanisms which mediate the enhanced proliferative potential of allylamine cells. Cellular growth and/or [3H]thymidine incorporation into DNA were evaluated in control and allylamine cells seeded on plastic culture dishes or glass coverslips in the presence of 0.1, 1 or 10% fetal bovine serum (FBS). On either substrate, incubation in 0.1% FBS for 48 h inhibited DNA synthesis in cultures of both cell types, but the inhibitory response was more pronounced in allylamine cells. Subsequent challenge with 10% FBS increased thymidine incorporation to a greater extent in allylamine cells. Interestingly, enhanced DNA synthesis of allylamine cells was associated with increased cell numbers only when seeded on a glass surface. The enhanced growth rate on glass was not due to increased plating efficiency since comparable attachment rates were observed for both cell types. Reseeding of control cells on glass substrates pre-coated by allylamine cells afforded control cells a growth advantage comparable with that observed for allylamine cultures. Conditioned media from growth-arrested, as well as cycling cultures, of allylamine cells stimulated DNA synthesis in cultures of either cell type to a greater extent than conditioned media from control counterparts. In addition, the responsiveness of allylamine cells to secreted products was enhanced relative to that of control cells. Metabolic labelling studies revealed that the synthesis and/or secretion of 52, 46, 33 and 28 kDa proteins was enhanced in allylamine cultures relative to controls, and that the expression of two proteins of 30 and 31 kDa only occurred in allylamine cultures. We conclude that the enhanced growth response of allylamine cells is associated with both altered protein secretion and differential extracellular matrix deposition.
Assuntos
Alilamina/farmacologia , Matriz Extracelular/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Animais , Aorta , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Células Cultivadas , Meios de Cultivo Condicionados , DNA/biossíntese , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Fenótipo , Ratos , Ratos Sprague-DawleyRESUMO
We have examined the ability of transforming growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor-BB (PDGF-BB) to regulate the expression of various integrins in cultured rabbit vascular smooth muscle cells (SMC). We found that expression of the alpha v beta 3 integrin complex was induced by both growth factors, although TGF-beta 1 appeared to be the more potent inducer. mRNA level of the beta 3 integrin subunit was undetectable in quiescent cells and enhanced by both growth factors, while the alpha v integrin subunit mRNA level did not change with growth factor addition. Therefore, appearance of the alpha v beta 3 integrin protein complex after growth factor stimulation was due to increased expression of the beta 3 integrin subunit mRNA. The TGF-beta 1 induced increase in beta 3 integrin mRNA was delayed, but did not require prior protein synthesis, since cycloheximide was unable to block the increase in beta 3 mRNA level. By contrast, PDGF-BB induced a more rapid increase in beta 3 integrin mRNA level that peaked by 6 h after growth factor addition and no detectable beta 3 integrin mRNA remained after 24 h. Interestingly, the PDGF-BB induced elevation of beta 3 integrin, although more rapid, was completely inhibited by cycloheximide. Expression of the alpha 5 integrin subunit in response to growth factors was very similar to beta 3. However, in contrast to beta 3 and alpha 5, neither TGF-beta 1 nor PDGF-BB were able to alter the expression of the beta 1 integrin subunit in vascular SMC. However, in TGF-beta 1 treated cells, there was a large increase in expression of a 190 kDa polypeptide that was associated with the beta 1 integrin subunit. This 190 kDa polypeptide was not detected in PDGF treated SMC or in TGF-beta 1 treated fibroblasts. The alpha 1 integrin subunit has a MW of approximately 190 kDa and is capable of complexing with beta 1. Analysis of the alpha 1 integrin subunit mRNA level indicated that it was indeed induced by TGF-beta 1, but not by PDGF-BB, suggesting that the 190 kDa polypeptide may be the alpha 1 integrin subunit. These results indicate that TGF-beta 1 and PDGF-BB are potent but distinct activators of integrin expression in vascular SMC.
Assuntos
Integrinas/biossíntese , Músculo Liso Vascular/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Becaplermina , Bovinos , Células Cultivadas , Integrinas/genética , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro/genética , Coelhos , SuínosRESUMO
We have studied the effect of transforming growth factor beta 1 (TGF-beta 1) on vascular smooth muscle cell (SMC) mitogenesis and expression of thrombospondin and other growth related genes. We found that TGF-beta 1 treatment of vascular SMC induced a prolonged increase in steady-state mRNA levels of thrombospondin as well as alpha 1 (IV) collagen. The increase began at approximately 2 h, peaked by 24 h, and remained considerably elevated 48 h after growth factor addition. There was a corresponding increase in thrombospondin protein as well as increased expression of several other secreted polypeptides. The increase in thrombospondin contrasted sharply with that observed for platelet-derived growth factor (PDGF) which induced a rapid and transient increase in thrombospondin mRNA level. Although TGF-beta 1 was able to directly enhance expression of thrombospondin as well as the growth-related genes c-fos and c-myc, and induced c-fos expression with identical kinetics as PDGF, it was unable to elicit [3H]thymidine incorporation into DNA in three independent smooth muscle cell strains. However, TGF-beta 1 was able to strongly increase the mitogenic response of SMC to PDGF. Addition of both TGF-beta 1 and PDGF to SMC also caused a synergistic increase in the expression of thrombospondin as well as c-myc. Interestingly, in one other smooth muscle cell strain, a weak and delayed mitogenic response to TGF-beta 1 alone was observed. Our results strongly suggest that induction of thrombospondin expression by TGF-beta 1 and by PDGF occurs by distinct mechanisms. In addition, that TGF-beta 1 can enhance PDGF-induced mitogenesis may be due to the ability of TGF-beta 1 to directly induce the expression of thrombospondin, c-fos, c-myc, and the PDGF beta-receptor.