RESUMEN
BACKGROUND: Brain oedema associated with cerebral contusion can be life-threatening. Mechanisms of the development of brain oedema are still unclear. METHOD: We investigated the expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (KDR/Flk-1) in the contusional brain tissue obtained during neurosurgery from 5 patients. FINDINGS: VEGF is expressed in some but not all the astrocytes, and KDR/Flk-1 is expressed in vascular endothelial cells in the con-tusional tissue as early as 3 hours after onset. CONCLUSION: The results suggested that the VEGF is induced in the contusional tissue in the very early period after onset, and that it increases capillary permeability via KDR/Flk-1 resulting in vasogenic type brain oedema.
Asunto(s)
Edema Encefálico/etiología , Edema Encefálico/metabolismo , Lesiones Encefálicas/complicaciones , Encéfalo/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adolescente , Adulto , Astrocitos/metabolismo , Lesiones Encefálicas/fisiopatología , Permeabilidad Capilar , Colorantes , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Eosina Amarillenta-(YS) , Femenino , Colorantes Fluorescentes , Hematoxilina , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Coloración y Etiquetado , Factores de Tiempo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
In humans, low peak bone mass is a significant risk factor for osteoporosis. We report that LRP5, encoding the low-density lipoprotein receptor-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG). We find that OPPG carriers have reduced bone mass when compared to age- and gender-matched controls. We demonstrate LRP5 expression by osteoblasts in situ and show that LRP5 can transduce Wnt signaling in vitro via the canonical pathway. We further show that a mutant-secreted form of LRP5 can reduce bone thickness in mouse calvarial explant cultures. These data indicate that Wnt-mediated signaling via LRP5 affects bone accrual during growth and is important for the establishment of peak bone mass.
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Densidad Ósea/genética , Anomalías del Ojo/genética , Ojo/embriología , Osteoblastos/metabolismo , Osteoporosis/genética , Receptores de LDL/fisiología , Factor de Crecimiento Transformador beta , Proteínas de Pez Cebra , Proteínas Adaptadoras Transductoras de Señales , Adulto , Animales , Animales no Consanguíneos , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/farmacología , Células COS , Niño , Preescolar , Chlorocebus aethiops , Cromosomas Humanos Par 11/genética , Medios de Cultivo Condicionados/farmacología , ADN Complementario/genética , Proteínas Dishevelled , Femenino , Genes Recesivos , Heterocigoto , Humanos , Proteínas Relacionadas con Receptor de LDL , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Masculino , Mesodermo/citología , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Fosfoproteínas/genética , Fosfoproteínas/fisiología , Proteínas/genética , Proteínas/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Receptores de LDL/deficiencia , Receptores de LDL/genética , Proteínas Recombinantes de Fusión/fisiología , Proteínas Recombinantes , Transducción de Señal , Cráneo/citología , Especificidad de la Especie , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Síndrome , Transfección , Proteínas Wnt , Proteína Wnt-5a , Proteína wnt2 , Proteína Wnt3 , Proteína Wnt4RESUMEN
PURPOSE: To localize endostatin and collagen type XVIII in human corneas and to characterize the enzymatic action of matrix metalloproteinases (MMPs) in the cleavage of collagen type XVIII and generation of endostatin in the cornea. METHODS: Anti-endostatin and anti-hinge antibodies were generated using peptide fragments corresponding to the endostatin region and the adjacent nonendostatin hinge region of collagen XVIII noncollagenous (NC)1 domain, respectively. Confocal immunostaining was performed to localize collagen XVIII in human corneas. SV40-immortalized corneal epithelial cells were immunoprecipitated and incubated with active MMP-1, -2, -3, -7, or -9, and Western blot analysis was performed to study collagen XVIII cleavage. Incubation with MMP-7 was performed at various concentrations (0, 2, 4, and 6 microg/ml) and time intervals (0, 1, 5, and 12 hours). Purified recombinant NC1 fragment of collagen XVIII was also digested with MMP-7, and the cleavage product was sequenced. RESULTS: Collagen XVIII was immunolocalized to the human corneal epithelium, epithelial basement membrane, and Descemet membrane. Western blot analysis demonstrated a 180- to 200-kDa band corresponding to collagen XVIII. MMP-7 (but not MMP-1, -2, -3, and -9) cleaved corneal epithelium-derived collagen XVIII to generate a 28-kDa endostatin-spanning fragment in a time- and concentration-dependent fashion. MMP-7 cleaved purified recombinant 34-kDa NC1 fragment of collagen XVIII in the hinge region to generate a 28-kDa fragment. CONCLUSIONS: Collagen XVIII is present in human cornea. MMP-7 cleaves the collagen XVIII NC1 domain to generate a 28-kDa fragment in the cornea.
Asunto(s)
Inhibidores de la Angiogénesis/metabolismo , Colágeno/metabolismo , Córnea/efectos de los fármacos , Metaloproteinasa 7 de la Matriz/farmacología , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/química , Animales , Formación de Anticuerpos , Membrana Basal/metabolismo , Western Blotting , Colágeno/química , Colágeno Tipo XVIII , Córnea/metabolismo , Lámina Limitante Posterior/metabolismo , Relación Dosis-Respuesta a Droga , Endostatinas , Epitelio Corneal/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Metaloproteinasa 7 de la Matriz/inmunología , Microscopía Confocal , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/química , ConejosRESUMEN
Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis during development, but little is known about the factors that control its expression. We provide the first example of tissue specific loss of VEGF expression as a result of targeting a single gene, Cbfa1/Runx2. During endochondral bone formation, invasion of blood vessels into cartilage is associated with upregulation of VEGF in hypertrophic chondrocytes and increased expression of VEGF receptors in the perichondrium. This upregulation is lacking in Cbfa1 deficient mice, and cartilage angiogenesis does not occur. Finally, over-expression of Cbfa1 in fibroblasts induces an increase in their VEGF mRNA level and protein production by stimulating VEGF transcription. The results demonstrate that Cbfa1 is a necessary component of a tissue specific genetic program that regulates VEGF during endochondral bone formation.
Asunto(s)
Huesos/embriología , Factores de Crecimiento Endotelial/genética , Regulación del Desarrollo de la Expresión Génica , Linfocinas/genética , Proteínas de Neoplasias , Osteogénesis , Factores de Transcripción/fisiología , Células 3T3 , Animales , Huesos/metabolismo , Cartílago/irrigación sanguínea , Cartílago/embriología , Cartílago/metabolismo , Condrocitos/citología , Condrocitos/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Factores de Crecimiento Endotelial/metabolismo , Marcación de Gen , Hibridación in Situ , Linfocinas/metabolismo , Ratones , Neovascularización Fisiológica , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tibia/embriología , Tibia/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Cherubism (MIM 118400) is an autosomal dominant inherited syndrome characterized by excessive bone degradation of the upper and lower jaws followed by development of fibrous tissue masses, which causes a characteristic facial swelling. Here we describe seven mutations in the SH3-binding protein SH3BP2 (MIM 602104) on chromosome 4p16.3 that cause cherubism.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Querubismo/genética , Mutación , Proteínas Portadoras/metabolismo , Querubismo/patología , Ligamiento Genético , Haplotipos/genética , Heterocigoto , Humanos , Linaje , Proteínas Proto-Oncogénicas c-abl/metabolismo , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
The angiogenesis inhibitor endostatin is a 20 kDA C-terminal fragment of collagen XVIII, a proteoglycan/collagen found in vessel walls and basement membranes. The endostatin fragment was originally identified in conditioned media from a murine endothelial tumor cell line. Endostatin inhibits endothelial cell migration in vitro and appears to be highly effective in murine in vivo studies. The molecular mechanisms behind the inhibition of angiogenesis have not yet been elucidated. Studies of the crystal structure of endostatin have shown a compact globular fold, with one face particularly rich in arginine residues acting as a heparin-binding epitope. It was initially suggested that zinc binding was essential for the antiangiogenic mechanism but later studies indicate that zinc has a structural rather than a functional role in endostatin. The generation of endostatin or endostatin-like collagen XVIII fragments is catalyzed by proteolytic enzymes, including cathepsin L and matrix metalloproteases, that cleave peptide bonds within the protease-sensitive hinge region of the C-terminal domain. The processing of collagen XVIII to endostatin may represent a local control mechanism for the regulation of angiogenesis.
Asunto(s)
Inhibidores de la Angiogénesis/metabolismo , Colágeno/química , Colágeno/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Inhibidores de la Angiogénesis/química , Animales , Embrión de Pollo , Colágeno Tipo XVIII , Endostatinas , Endotelio Vascular/metabolismo , Humanos , Ratones , Péptido Hidrolasas/metabolismoRESUMEN
BACKGROUND: Malignant tumours may produce substances with both stimulatory and inhibitory effect on angiogenesis. MATERIAL AND METHODS: A protein fragment with angiogenesis-inhibiting potential was recently identified in conditioned media from a murine endothelial tumour cell line. RESULTS: The angiogenesis inhibitor, endostatin, is a 20 kDa C-terminal fragment of collagen XVIII, a proteoglycan/collagen found in vessel walls and basement membranes. The generation of endostatin or endostatin-like collagen XVIII fragments is catalyzed by proteolytic enzymes, including cathepsin L and matrix metalloproteases, that cleave peptide bonds within the protease-sensitive hinge region of the C-terminal domain. INTERPRETATION: The physiological processing of collagen XVIII to endostatin may represent a local control mechanism for the regulation of angiogenesis. The outcome of ongoing clinical trials will determine the role of endostatin as a possible angiogenesis-inhibiting drug in the future.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Colágeno/química , Neoplasias/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Fragmentos de Péptidos/administración & dosificación , Inhibidores de la Angiogénesis/química , Animales , Factores de Crecimiento Endotelial/administración & dosificación , Factores de Crecimiento Endotelial/química , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Fragmentos de Péptidos/química , Células Tumorales CultivadasRESUMEN
Moyamoya disease is a progressive cerebrovascular occlusive disease that primarily affects children. The cause is unknown. We examined the production of prostanoids and the expression of cyclooxygenase-2 (COX-2) in cultured arterial smooth muscle cells (SMCs) derived from patients with moyamoya disease. Twelve moyamoya and 8 control cell strains were examined. The steady-state levels of prostanoids in the culture medium did not differ between moyamoya and control SMCs. When the cells were stimulated with interleukin-1beta (IL-1beta), prostaglandin E(2) (PGE(2)) release into the medium was significantly greater from moyamoya SMCs than from control SMCs, whereas the amounts of prostacyclin and thromboxane B(2) did not differ. IL-1beta-induced PGE(2) production by moyamoya SMCs was completely blocked by the addition of indomethacin or NS-398. IL-1beta significantly stimulated cell migration and DNA synthesis in control SMCs but had an inhibitory effect on moyamoya SMCs. The inhibitory effects on the growth and migration of moyamoya SMCs were caused by excessive secretion of PGE(2) and was reversed with indomethacin treatment. Immunofluorescence studies and Western blot analysis showed greater amounts of COX-2 protein expression in IL-1beta-stimulated moyamoya SMCs. These findings suggest that moyamoya SMCs respond to inflammatory stimuli to produce excess amounts of PGE(2) through the activation of COX-2, which increases vascular permeability and decreases vascular tone. This facilitates the exposure of vessels to blood constituents and promotes the development of intimal thickening in moyamoya disease.
Asunto(s)
Dinoprostona/biosíntesis , Interleucina-1/farmacología , Enfermedad de Moyamoya/metabolismo , Músculo Liso Vascular/metabolismo , Adolescente , Arterias/metabolismo , Arterias/patología , Arterias/fisiopatología , Movimiento Celular , Células Cultivadas , Niño , Preescolar , Medios de Cultivo/metabolismo , Ciclooxigenasa 2 , ADN/biosíntesis , Femenino , Humanos , Lactante , Isoenzimas/metabolismo , Masculino , Proteínas de la Membrana , Enfermedad de Moyamoya/patología , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Prostaglandinas/biosíntesisRESUMEN
Endostatin, produced as recombinant protein in human 293-EBNA cells, inhibits the migration of human umbilical vein endothelial cells (HUVECs) in response to vascular endothelial growth factor (VEGF) in a dose-dependent manner and prevents the subcutaneous growth of human renal cell carcinomas in nude mice at concentrations and in doses that are from 1000- to 100 000-fold lower than those previously reported. The inhibition of migration is not affected by mutations which eliminate Zn or heparin binding and inhibition of tumor growth does not depend on Zn binding. The results of the migration assays suggest that endostatin causes a block at one or more steps in VEGF-induced migration, while VEGF in turn can cause a block of the inhibition by endostatin of VEGF-induced migration of HUVECs.
Asunto(s)
Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Colágeno/farmacología , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/efectos de los fármacos , Linfocinas/farmacología , Fragmentos de Péptidos/farmacología , Animales , Antineoplásicos/aislamiento & purificación , Antineoplásicos/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/fisiopatología , Línea Celular Transformada , Células Cultivadas , Colágeno/genética , Colágeno/aislamiento & purificación , Colágeno/metabolismo , Endostatinas , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Endotelio Vascular/citología , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/fisiopatología , Linfocinas/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutagénesis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , ZincRESUMEN
Cherubism is an autosomal dominant disorder that may be related to tooth development and eruption. It is a disorder of age-related bone remodeling, mostly limited to the maxilla and the mandible, with loss of bone in the jaws and its replacement with large amounts of fibrous tissue. We have used a genomewide search with a three-generation family and have established linkage to chromosome 4p16. Three other families affected with cherubism were also genotyped and were mapped to the same locus. The combined LOD score is 4.21 at a recombination fraction of 0, and the locus spans an interval of approximately 22 cM.
Asunto(s)
Querubismo/genética , Cromosomas Humanos Par 4 , Adolescente , Adulto , Querubismo/diagnóstico , Querubismo/diagnóstico por imagen , Niño , Mapeo Cromosómico , Femenino , Marcadores Genéticos , Genotipo , Humanos , Escala de Lod , Masculino , Mandíbula/diagnóstico por imagen , Maxilar/anatomía & histología , Maxilar/diagnóstico por imagen , Persona de Mediana Edad , Linaje , RadiografíaRESUMEN
The C-terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non-covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25-32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N-terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin-1 and fibulin-2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin-1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22-38 kDa) and in serum (120-300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N-terminal association region (approximately 50 residues), a central protease-sensitive hinge region (approximately 70 residues) and a C-terminal stable endostatin domain (approximately 180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix-associated to a more soluble endocrine form.
Asunto(s)
Colágeno/química , Neovascularización Patológica/prevención & control , Fragmentos de Péptidos/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Metabolismo de los Hidratos de Carbono , Línea Celular , Colágeno/biosíntesis , Colágeno/sangre , Colágeno/genética , Colágeno/metabolismo , Colágeno Tipo XVIII , Endopeptidasas , Endostatinas , Vectores Genéticos , Humanos , Hidrólisis , Ligandos , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
BACKGROUND AND PURPOSE: Moyamoya disease is a progressive cerebrovascular occlusive disease affecting primarily children. The etiology remains unknown. We examined the chemotactic and proliferative activities of inflammatory cell products from arterial smooth muscle cells (SMCs) derived from moyamoya patients and compared them with those from control subjects. METHODS: We used 12 SMC strains from moyamoya patients and eight from control subjects. SMC migration was examined in a micro chemotaxis chamber. DNA synthesis was measured by an immunoperoxidase technique. RESULTS: Platelet-derived growth factor (PDGF)-BB markedly stimulated cell migration and DNA synthesis in control SMCs. PDGF-AA stimulated only DNA synthesis in control SMCs. In moyamoya SMCs, PDGF-AA and PDGF-BB stimulated cell migration but not DNA synthesis. Basic fibroblast growth factor had little migratory activity but stimulated DNA synthesis in moyamoya SMCs and control SMCs. Conversely, hepatocyte growth factor stimulated cell migration but not DNA synthesis in moyamoya SMCs and control SMCs. In contrast, interleukin-1 beta (IL-1 beta) significantly stimulated the migration and DNA synthesis of control SMCs, while it inhibited moyamoya SMC migration. The levels of IL-1 beta-induced nitric oxide production did not differ between moyamoya SMCs and control SMCs, suggesting that IL-1 beta inhibits the migration of moyamoya SMCs through a nitric oxide-independent pathway. CONCLUSIONS: The differences in responses to PDGF and IL-1 in moyamoya SMCs are involved in the mechanism by which intimal thickening develops in moyamoya disease.
Asunto(s)
Mitógenos/farmacología , Enfermedad de Moyamoya/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Anticoagulantes/farmacología , Becaplermina , Bromodesoxiuridina , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , ADN/metabolismo , Humanos , Óxido Nítrico/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-sis , Proteínas Recombinantes/farmacologíaRESUMEN
To investigate the possible mechanism of neointimal formation in Moyamoya disease, we histologically examined the superficial temporal arteries and also investigated cultured smooth muscle cells (SMCs) from the arteries. Intimal thickening of the scalp arteries developed significantly at an early age in Moyamoya patients compared with control subjects. The histopathological findings of the neointima in scalp arteries were almost similar to those in intracranial arteries in Moyamoya patients. SMCs cultured from Moyamoya arteries responded significantly less to serum mitogens, especially to platelet derived growth factor (PDGF), than those of control patients, the finding of which was explained by the reduced number of PDGF receptor on Moyamoya SMCs. Our findings indicate the presence of systemic factors that promote migration and proliferation of SMCs from the media to the intima in Moyamoya disease. Our results suggest that alteration in vascular cells may contribute to the development of intimal thickening in Moyamoya disease.
Asunto(s)
Enfermedad de Moyamoya/patología , Arterias Temporales/patología , Túnica Íntima/patología , Adolescente , Adulto , Técnicas de Cultivo de Célula , Movimiento Celular , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Músculo Liso/patología , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
The matrix metalloproteinases (MMPs) are a family of zinc-containing matrix degrading endopeptidases. A subfamily of membrane type (MT) -MMPs has been described recently. We have determined the structure of the gene (Mmp14) encoding the first MT-MMP to be described, MT1-MMP (MMP-14), and mapped it to mouse chromosome 14. The mouse MMP-14 protein is encoded by ten exons. The novel C-terminal peptide domains of MMP-14 are encoded by a single large exon that also encodes the 3'-untranslated region. The structure of the exons encoding the catalytic domain and pro-domain of MMP-14 is distinct from previously described MMP genes, whereas the exons encoding the hemopexin-like domains are similar to those of most other MMP genes. Mmp14 and the gene for tissue inhibitor of metalloproteinases-2 (Timp2) show a temporally and spatially co-regulated expression during mouse development. They are co-expressed during vascular and urogenital development and during the development of osteocartilaginous and musculotendinous structures. The stringent co-expression of these two genes suggests common regulatory pathways that may have important functional implications for the activation of pro-gelatinase A in health and disease.
Asunto(s)
Embrión de Mamíferos/enzimología , Metaloendopeptidasas/genética , Inhibidores de Proteasas/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/genética , Animales , Sitios de Unión , Northern Blotting , Huesos/embriología , Huesos/enzimología , Mapeo Cromosómico , Cromosomas , Desarrollo Embrionario y Fetal , Exones , Expresión Génica , Intrones , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Ratones , Datos de Secuencia Molecular , Músculos/embriología , Músculos/enzimología , Análisis de Secuencia de ADNRESUMEN
BACKGROUND AND PURPOSE: Moyamoya disease is a progressive cerebrovascular occlusive disease that is rare in all ages but frequently presents in children. The etiology of the disease is unknown. We examined elastin gene transcripts and elastin synthesis in cultured arterial smooth muscle cells (SMCs) derived from moyamoya patients and compared them with those in SMCs from age-matched control subjects. METHODS: We used six cell strains from moyamoya patients and four from controls. The expression of elastin protein was observed by Western blot analysis and metabolic labeling with 3H-valine. Elastin gene transcripts were identified by Northern blot analysis. RESULTS: Elastin mRNA and protein levels were elevated in all SMCs from moyamoya patients compared with control SMCs. Although transforming growth factor-beta 1 (TGF-beta 1), a potent enhancer of the expression of elastin in arterial SMCs, upregulated elastin mRNA and protein levels in SMCs from both moyamoya patients and control subjects, the maximum levels of elastin synthesis and elastin gene transcripts in response to exogenous TGF-beta 1 were significantly greater in moyamoya SMCs than control SMCs. In addition, quiescent moyamoya SMCs secreted significantly more TGF-beta 1 into the culture medium than quiescent control SMCs (P < .01). CONCLUSIONS: Our findings suggest that moyamoya disease may result, at least in part, from an abnormal regulation of extracellular matrix metabolism that leads to increased steady state levels of elastin mRNA and elastin accumulation in the intimal thickening and that increased elastin accumulation is a stable marker of SMCs from patients with moyamoya disease.
Asunto(s)
Arterias/fisiopatología , Elastina/genética , Elastina/metabolismo , Expresión Génica , Enfermedad de Moyamoya/genética , Enfermedad de Moyamoya/metabolismo , Músculo Liso/fisiopatología , Adolescente , Arterias/patología , Northern Blotting , Western Blotting , Células Cultivadas , Niño , Preescolar , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Masculino , Enfermedad de Moyamoya/patología , Músculo Liso/patología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Cleidocranial dysplasia (CCD) is an autosomal dominant disease characterized by hypoplasia or aplasia of clavicles, open fontanelles, and other skeletal anomalies. A mouse mutant, shown by clinical and radiographic analysis to be strikingly similar to the human disorder and designated Ccd, was used as a model for the human disorder. Since malformation of the clavicle is the hallmark of CCD, we studied clavicular development in wild-type and Ccd mice. Histology and in situ hybridization experiments were performed to compare the temporal and spatial expression of several genes in wild-type and Ccd mutant mouse embryos. Bone and cartilage specific markers--type I, II, and X collagens, Sox9, aggrecan, and osteopontin were used as probes. The analyses covered the development of the clavicle from the initial mesenchymal condensation at embryonic day 13 (E13) to the late mineralization stage at embryonic day 15.5. At day 13.5, cells in the center of the condensation differentiate into characteristic precursor cells that were not observed in other bone anlagen. In the medial part of the anlage these cells express markers of the early cartilage lineage (type II collagen and Sox9), whereas cells of the lateral part express markers of the osteoblast lineage (type I collagen). With further development the medial cells differentiate into chondrocytes and start to express chondrocyte-specific markers such as aggrecan. Cells of the lateral part differentiate into osteoblasts as indicated by the production of bone matrix and the expression of osteopontin. At day 14.5 a regular growth plate has developed between the two parts where type X collagen expression can be demonstrated in hypertrophic chondrocytes. The data indicate that the medial part of the clavicle develops by endochondral bone formation while the lateral part ossifies as a membranous bone. The clavicle of Ccd mice showed a smaller band of mesenchymal cell condensation than in wild-type mice. Cells of the condensation failed to express type I and type II collagen at E13.5. In the lateral part of the clavicle type I collagen expression was not detected until E14.5 and osteopontin expression only appeared at E15.5. At E15.5, a small ossification center appears in the lateral part which is, in contrast to the wild-type clavicular bone, solid and without primary spongiosa as well as bone marrow. In the medial portion, type II collagen expression and endochondral ossification never occurs in Ccd mice; this portion of the clavicle is therefore missing in Ccd.
Asunto(s)
Clavícula/anomalías , Displasia Cleidocraneal/patología , Animales , Huesos/metabolismo , Cartílago/metabolismo , Colágeno/metabolismo , Hibridación in Situ , Mesodermo/metabolismo , Mesodermo/patología , Ratones , Ratones Mutantes , Osteopontina , Sialoglicoproteínas/metabolismoRESUMEN
We previously identified the angiogenesis inhibitor angiostatin. Using a similar strategy, we have identified endostatin, an angiogenesis inhibitor produced by hemangioendothelioma. Endostatin is a 20 kDa C-terminal fragment of collagen XVIII. Endostatin specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth. By a novel method of sustained release, E. coli-derived endostatin was administered as a nonrefolded suspension. Primary tumors were regressed to dormant microscopic lesions. Immunohistochemistry revealed blocked angiogenesis accompanied by high proliferation balanced by apoptosis in tumor cells. There was no toxicity. Together with angiostatin data, these findings validate a strategy for identifying endogenous angiogenesis inhibitors, suggest a theme of fragments of proteins as angiogenesis inhibitors, and demonstrate dormancy therapy.
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Antineoplásicos/farmacología , Colágeno/farmacología , Hemangioendotelioma/metabolismo , Neoplasias Experimentales/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/uso terapéutico , Capilares/citología , División Celular/efectos de los fármacos , Colágeno/química , Colágeno/aislamiento & purificación , Colágeno/toxicidad , Colágeno Tipo XVIII , Medios de Cultivo Condicionados , Endostatinas , Endotelio Vascular/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/patología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/toxicidad , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Células Tumorales CultivadasRESUMEN
BACKGROUND AND PURPOSE: Moyamoya disease is a progressive cerebrovascular occlusive disease that occurs in children. The etiology is unknown. We examined the superficial temporal arteries from patients with moyamoya disease, particularly children, to determine whether the extracranial arteries as well as the intracranial arteries are involved in this disease. METHODS: Small branches of the superficial temporal arteries were obtained from 22 patients with moyamoya disease during indirect arterial bypass surgery. Histological examinations were performed, and the findings were compared with those of arteries from 12 control patients. RESULTS: Intimal thickening was observed in 9 of 17 patients with moyamoya disease younger than 20 years but in none of 7 control patients under the age of 20 years (P < .02, Fisher's exact test). Intimal thickening appeared from age 20 years in control patients. The arteries of moyamoya patients showed fibrocellular intimal thickening with a paucity of lipid. The arteries from moyamoya patients contained strongly stained multilayered elastic fibers in the thickened intima, while those from control patients showed only weakly stained elastic fibers in the intima. CONCLUSIONS: Our findings suggest that moyamoya disease is a systemic vascular disease. The results indicate systemic etiologic factors that may promote the early development of intimal thickening in moyamoya disease.
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Enfermedad de Moyamoya/patología , Arterias Temporales/patología , Túnica Íntima/patología , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de TiempoRESUMEN
Platelet-derived growth factor (PDGF) is one of the major mitogens in serum to stimulate replication of human smooth muscle cells (SMCs) in culture. Previous studies using human fibroblasts failed to demonstrate changes in the receptor systems for growth factors during cellular senescence. We investigated the kinetics of 125I-PDGF(-BB) binding and down-regulation of the PDGF receptor in three human arterial SMC strains during cellular aging. The number of specific 125I-PDGF binding sites per cell increased slightly at a population doubling level (PDL) of 60%-80% of life span and then decreased at the PDL above 90%. The number of receptors per cell-surface area decreased with increasing in vitro age. The apparent Kd for the 125I-PDGF binding decreased with in vitro senescence. The internalization and degradation of 125I-PDGF per receptor were significantly reduced in senescent SMCs and the amount of 125I-PDGF that escaped degradation and was recycled back to the cell surface was significantly greater in senescent SMCs than young cells. Furthermore, down-regulation of the PDGF receptor was significantly greater in senescent SMCs than young cells. Immunoblot studies demonstrated that changes in beta-subunit of the PDGF receptor accounted for those in the studies using 125I-PDGF and that tyrosine phosphorylation of the PDGF receptor was significantly greater in young SMCs than aged cells. Our results suggest that age-related changes in the receptor systems for PDGF may be important contributors to the failure of DNA synthesis in senescent SMCs.
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Arterias/metabolismo , Regulación hacia Abajo , Músculo Liso Vascular/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Arterias/citología , Western Blotting , Células Cultivadas , Senescencia Celular , ADN/biosíntesis , Humanos , Radioisótopos de Yodo , Cinética , Músculo Liso Vascular/citología , Fosforilación , Tirosina/metabolismoRESUMEN
A three-dimensional corneal tissue construct was used to examine the effect of culture environment and endothelial cell interaction on epithelial differentiation and basement membrane assembly. Rabbit corneal epithelial cells were cultured over rabbit stromal fibroblasts in a collagen matrix with or without an underlying layer of immortalized mouse corneal endothelial cells (Muragaki, Shiota, Inoue, Ooshima, Olsen, and Ninomiya. (1992) Eur. J. Biochem. 207, 895-902). The cultures were grown submerged or at a dry or moist interface. Basement membrane, anchoring fibril, and hemidesmosome assembly was monitored using transmission electron microscopy as well as indirect immunofluorescence microscopy of laminin, type VII collagen, and alpha 6 integrin. Antibodies against keratin 3 (K3) and alpha-enolase marked differentiated and undifferentiated corneal epithelial cells, respectively. When all three cell types were cultured at a moist interface, hemidesmosomes, anchoring fibrils, and a continuous basement membrane were observed 2 wk after lifting the cultures to an air-liquid interface (air-lift). The distribution of alpha-enolase and K3 was identical to patterns seen in the limbal region of the cornea. Air-lifted tissue constructs lacking the endothelial cell layer showed only limited distribution of laminin and type VII collagen at the epithelial-matrix junction. alpha 6 Integrin was present along the entire plasma membrane of the basal cells; epithelial differentiation was not complete as alpha-enolase was seen in basal and two to three layers of suprabasal cells. Submerged cultures without endothelial cells did not express differentiation markers or basement membrane components. These data indicate that endothelial cell interaction dramatically enhances the amount and quality of epithelial basement membrane assembly and that epithelial differentiation is influenced by the type of interface between tissue, liquid, and air.