RESUMEN
The molecular mechanisms predisposing to atherosclerotic aneurysm formation remain undefined. Nevertheless, rupture of aortic aneurysms is a major cause of death in Western societies, with few available treatments and poor long-term prognosis. Indirect evidence suggests that matrix metalloproteinases (MMPs) and plasminogen activators (PAs) are involved in its pathogenesis. MMPs are secreted as inactive zymogens (pro-MMPs), requiring activation in the extracellular compartment. Plasmin, generated from the zymogen plasminogen by tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA; refs 14,15), has been proposed as a possible activator in vitro, but evidence for such a role in vivo is lacking. Analysis of atherosclerotic aorta in mice with a deficiency of apoliprotein E (Apoe-/-; ref. 18), singly or combined with a deficiency of t-PA (Apoe-/-:Plat-/-) or of u-PA (Apoe-/-:Plau-/-; ref. 19), indicated that deficiency of u-PA protected against media destruction and aneurysm formation, probably by means of reduced plasmin-dependent activation of pro-MMPs. This genetic evidence suggests that plasmin is a pathophysiologically significant activator of pro-MMPs in vivo and may have implications for the design of therapeutic strategies to prevent aortic-wall destruction by controlling Plau gene function.
Asunto(s)
Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Torácica/enzimología , Fibrinolisina/metabolismo , Metaloendopeptidasas/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Aneurisma de la Aorta Abdominal/etiología , Aneurisma de la Aorta Abdominal/patología , Aneurisma de la Aorta Torácica/etiología , Aneurisma de la Aorta Torácica/patología , Arteriosclerosis/enzimología , Arteriosclerosis/patología , Colágeno/metabolismo , Dieta Aterogénica , Elastina/metabolismo , Activación Enzimática , Femenino , Macrófagos/enzimología , Masculino , Ratones , Ratones Noqueados , Túnica Media/enzimología , Túnica Media/patologíaRESUMEN
Hypoxia stimulates angiogenesis through the binding of hypoxia-inducible factors to the hypoxia-response element in the vascular endothelial growth factor (Vegf) promotor. Here, we report that deletion of the hypoxia-response element in the Vegf promotor reduced hypoxic Vegf expression in the spinal cord and caused adult-onset progressive motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis. The neurodegeneration seemed to be due to reduced neural vascular perfusion. In addition, Vegf165 promoted survival of motor neurons during hypoxia through binding to Vegf receptor 2 and neuropilin 1. Acute ischemia is known to cause nonselective neuronal death. Our results indicate that chronic vascular insufficiency and, possibly, insufficient Vegf-dependent neuroprotection lead to the select degeneration of motor neurons.
Asunto(s)
Hipoxia de la Célula/genética , Factores de Crecimiento Endotelial/genética , Linfocinas/genética , Neuronas Motoras/patología , Degeneración Nerviosa/genética , Elementos de Respuesta/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Axones/fisiología , Sitios de Unión , Electrofisiología , Factores de Crecimiento Endotelial/metabolismo , Humanos , Linfocinas/metabolismo , Ratones , Ratones Noqueados , Neuronas Motoras/fisiología , Contracción Muscular , Fibras Musculares Esqueléticas/patología , Atrofia Muscular/genética , Atrofia Muscular/patología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuropilina-1 , Nervios Periféricos/patología , Regiones Promotoras Genéticas , 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 , Eliminación de Secuencia , Médula Espinal/fisiología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The growth arrest-specific gene 6 product (Gas6) is a secreted protein related to the anticoagulant protein S but its role in hemostasis is unknown. Here we show that inactivation of the Gas6 gene prevented venous and arterial thrombosis in mice, and protected against fatal collagen/epinephrine-induced thrombo embolism. Gas6-/- mice did not, however, suffer spontaneous bleeding and had normal bleeding after tail clipping. In addition, we found that Gas6 antibodies inhibited platelet aggregation in vitro and protected mice against fatal thrombo embolism without causing bleeding in vivo. Gas6 amplified platelet aggregation and secretion in response to known agonists. Platelet dysfunction in Gas6-/- mice resembled that of patients with platelet signaling transduction defects. Thus, Gas6 is a platelet-response amplifier that plays a significant role in thrombosis. These findings warrant further evaluation of the possible therapeutic use of Gas6 inhibition for prevention of thrombosis.
Asunto(s)
Plaquetas/fisiología , Péptidos y Proteínas de Señalización Intercelular , Proteínas/fisiología , Trombosis/prevención & control , Animales , Plaquetas/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Hemostasis , Humanos , Masculino , Ratones , Ratones Noqueados , Fenotipo , Agregación Plaquetaria , Proteínas/genética , Proteínas/inmunología , Proteínas/farmacología , Receptores de Superficie Celular/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Trombosis/etiologíaRESUMEN
Cardiac rupture is a fatal complication of acute myocardial infarction lacking treatment. Here, acute myocardial infarction resulted in rupture in wild-type mice and in mice lacking tissue-type plasminogen activator, urokinase receptor, matrix metalloproteinase stromelysin-1 or metalloelastase. Instead, deficiency of urokinase-type plasminogen activator (u-PA-/-) completely protected against rupture, whereas lack of gelatinase-B partially protected against rupture. However, u-PA-/- mice showed impaired scar formation and infarct revascularization, even after treatment with vascular endothelial growth factor, and died of cardiac failure due to depressed contractility, arrhythmias and ischemia. Temporary administration of PA inhibitor-1 or the matrix metalloproteinase-inhibitor TIMP-1 completely protected wild-type mice against rupture but did not abort infarct healing, thus constituting a new approach to prevent cardiac rupture after acute myocardial infarction.
Asunto(s)
Gasto Cardíaco Bajo/etiología , Rotura Cardíaca/etiología , Metaloendopeptidasas/antagonistas & inhibidores , Infarto del Miocardio/complicaciones , Infarto del Miocardio/tratamiento farmacológico , Inactivadores Plasminogénicos/uso terapéutico , Inhibidores de Proteasas/uso terapéutico , Animales , Arritmias Cardíacas , Trasplante de Médula Ósea , Movimiento Celular , Colagenasas/metabolismo , Técnicas de Transferencia de Gen , Leucocitos/citología , Leucocitos/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 9 de la Matriz , Ratones , Ratones Mutantes , Neovascularización Fisiológica/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Activadores Plasminogénicos/genética , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismoRESUMEN
We have tested the hypothesis that phenotypic changes of development are accompanied by expression of differentiation-stage specific peptides bound to major histocompatibility complex (MHC) class I molecules. The U937 cell line, when cultured in the presence of phorbol myristate acetate (PMA), undergoes differentiation from monoblasts to macrophage-like cells. The high-performance liquid chromatography profile of peptides eluted from purified human histocompatibility leukocyte antigen class I molecules expressed by U937 treated with PMA differs from that obtained from control, untreated U937 cells. Chemical sequencing of eluted peptides identified a peptide derived from cytomegalovirus in both treated and untreated cells. PMA-treated, but not untreated cells, displayed an additional peptide derived from interleukin 1 beta. Hence, differentiation-induction of U937 is accompanied by the presentation of at least one differentiation-stage specific peptide. Our results indicate that, similar to viral infection, cellular development and transformation is accompanied by the de novo synthesis of proteins which are processed and presented on MHC class I molecules.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Leucemia Mieloide/patología , Complejo Mayor de Histocompatibilidad/inmunología , Péptidos/metabolismo , Secuencia de Aminoácidos , Diferenciación Celular , Transformación Celular Neoplásica/patología , Cromatografía Líquida de Alta Presión , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Leucemia Mieloide/inmunología , Leucemia Mieloide/metabolismo , Datos de Secuencia Molecular , Péptidos/análisis , Fenotipo , Pruebas de Precipitina , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Inflammation drives arterial, venous and microvascular thrombosis. Chronic inflammation contributes to arterial thrombotic complications, whereas acute inflammation drives venous thrombosis and microvascular thrombosis. Mechanistically, inflammation modulates thrombotic responses by upregulating procoagulants, downregulating anticoagulants and suppressing fibrinolysis. The inflammatory response can also result in cell apoptosis or necrosis. Products released from the dead cells, particularly histones, propagate further inflammation, tissue death and organ failure. Inhibition of histone mediated cytotoxicity appears to be a new mechanism for protecting against this deadly cascade.
Asunto(s)
Coagulación Sanguínea/fisiología , Inmunidad Innata , Inflamación/fisiopatología , Anticoagulantes/farmacología , Coagulación Sanguínea/inmunología , Muerte Celular , Fibrinólisis/fisiología , Histonas/antagonistas & inhibidores , Humanos , Inflamación/complicaciones , Inflamación/inmunología , Inflamación/patología , Inhibidores de Agregación Plaquetaria/farmacología , Trombosis/etiología , Trombosis/fisiopatologíaRESUMEN
We investigated the distribution of sterols in the cell membrane of microvascular endothelium (mouse pancreas, diaphragm, brain, heart, lung, kidney, thyroid, adrenal, and liver) with the polyene antibiotic filipin, which reportedly has binding specificity for free 3-beta-hydroxysterols. In some experiments, concomitantly, cell-surface anionic sites were detected with cationized ferritin. Vessels were perfused in situ with PBS, followed by light fixation and filipin administration for 10 to 60 min. Tissues were further processed for thin-section and freeze-fracture electron microscopy. Short exposure (10 min) to filipin-glutaraldehyde solution resulted in the initial appearance, on many areas, of rings of characteristic filipin-sterol complexes within the rim surrounding stomata of most plasmalemmal vesicles, transendothelial channels, and fenestrae. Such rings were absent from the rims of the large openings of the sinusoid endothelium (liver, adrenal), coated pits and phagocytic vacuoles. After longer exposure (30-60 min), filipin-sterol complexes labeled randomly the rest of plasma membrane (except for coated pits, and partially the interstrand areas of junctions), and also marked most plasmalemmal vesicles. These peristomal rings of sterols were displayed mostly on the P face, and, at their full development, consisted of 6-8 units around a vesicle stoma, and 10-12 units around a fenestra. At their level, the intramembranous particles and the cell surface anionic sites were virtually excluded. Peristomal rings of sterols were also detected on the plasma membrane of pericytes and smooth muscle cells of the microvascular wall, which otherwise were poorly labeled with filipin-sterol complexes as compared to endothelial plasmalemma. It is presumed that the peristomal rings of cholesterol may represent important contributors to the local transient stabilization of plasma membrane and to the phase separation between cell membrane and vesicle membrane at a certain stage of their fusion/fission process.
Asunto(s)
Capilares/ultraestructura , Esteroles/análisis , Animales , Membrana Celular/ultraestructura , Endotelio/ultraestructura , Filipina , Técnica de Fractura por Congelación , Glutaral , Masculino , Ratones , Páncreas/irrigación sanguínea , Ratas , Glándula Tiroides/irrigación sanguínea , Factores de TiempoRESUMEN
In previous studies we have shown that, after stimulation by a receptor ligand such as thrombin, tissue-type plasminogen activator (tPA) and von Willebrand factor (vWf) will be acutely released from human umbilical vein endothelial cells (HUVEC). However, the mechanisms involved in the secretion of these two proteins differ in some respects, suggesting that the two proteins may be stored in different secretory granules. By density gradient centrifugation of rat lung homogenates, a particle was identified that contained nearly all tPA activity and antigen. This particle had an average density of 1.11-1.12 g/ml, both in Nycodenz density gradients and in sucrose density gradients. A similar density distribution of tPA was found for a rat endothelial cell line and for HUVEC. After thrombin stimulation of HUVEC to induce tPA secretion, the amount of tPA present in high-density fractions decreased, concomitant with the release of tPA into the culture medium and a shift in the density distribution of P-selectin. vWf, known to be stored in Weibel-Palade bodies, showed an identical distribution to tPA in Nycodenz gradients. In contrast, the distribution in sucrose gradients of vWf from both rat and human lung was very different from that of tPA, suggesting that tPA and vWf were not present in the same particle. Using double-immunofluorescence staining of HUVEC, tPA- and vWf-containing particles showed a different distribution by confocal microscopy. The distribution of tPA also differed from the distribution of tissue factor pathway inhibitor, endothelin-1, and caveolin. By immunoelectronmicroscopy, immunoreactive tPA could be demonstrated in small vesicles morphologically different from the larger Weibel-Palade bodies. It is concluded that tPA in endothelial cells is stored in a not-previously-described, small and dense (d = 1.11-1.12 g/ml) vesicle, which is different from a Weibel-Palade body.
Asunto(s)
Caveolinas , Gránulos Citoplasmáticos/enzimología , Endotelio Vascular/enzimología , ATPasas de Translocación de Protón , Proteínas de Saccharomyces cerevisiae , Activador de Tejido Plasminógeno/metabolismo , Animales , Caveolina 1 , Línea Celular , Centrifugación por Gradiente de Densidad/métodos , Endotelina-1/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Inmunohistoquímica , Yohexol , Pulmón/enzimología , Proteínas de la Membrana/metabolismo , Ratones , Ratas , Sacarosa , Trombina/farmacología , Activador de Tejido Plasminógeno/efectos de los fármacos , Factor de von Willebrand/metabolismoRESUMEN
It has been proposed that the urokinase receptor (u-PAR) is essential for the various biological roles of urokinase-type plasminogen activator (u-PA) in vivo, and that smooth muscle cells require u-PA for migration during arterial neointima formation. The present study was undertaken to evaluate the role of u-PAR during this process in mice with targeted disruption of the u-PAR gene (u-PAR-/-). Surprisingly, u-PAR deficiency did not affect arterial neointima formation, neointimal cell accumulation, or migration of smooth muscle cells. Indeed, topographic analysis of arterial wound healing after electric injury revealed that u-PAR-/- smooth muscle cells, originating from the uninjured borders, migrated over a similar distance and at a similar rate into the necrotic center of the wound as wild-type (u-PAR+/+) smooth muscle cells. In addition, u-PAR deficiency did not impair migration of wounded cultured smooth muscle cells in vitro. There were no genotypic differences in reendothelialization of the vascular wound. The minimal role of u-PAR in smooth muscle cell migration was not because of absent expression, since wild-type smooth muscle cells expressed u-PAR mRNA and functional receptor in vitro and in vivo. Pericellular plasmin proteolysis, evaluated by degradation of 125I-labeled fibrin and activation of zymogen matrix metalloproteinases, was similar for u-PAR-/- and u-PAR+/+ cells. Immunoelectron microscopy of injured arteries in vivo revealed that u-PA was bound on the cell surface of u-PAR+/+ cells, whereas it was present in the pericellular space around u-PAR-/- cells. Taken together, these results suggest that binding of u-PA to u-PAR is not required to provide sufficient pericellular u-PA-mediated plasmin proteolysis to allow cellular migration into a vascular wound.
Asunto(s)
Arteria Femoral/fisiología , Fibrinolisina/metabolismo , Metaloendopeptidasas/metabolismo , Músculo Liso Vascular/fisiología , Receptores de Superficie Celular/fisiología , Túnica Íntima/fisiología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Cicatrización de Heridas/fisiología , Animales , Células Cultivadas , Quimiotaxis/genética , Quimiotaxis/fisiología , Femenino , Arteria Femoral/citología , Arteria Femoral/lesiones , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Microscopía Inmunoelectrónica , Músculo Liso Vascular/citología , Músculo Liso Vascular/lesiones , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Regeneración , Túnica Íntima/citología , Túnica Íntima/lesiones , Cicatrización de Heridas/genéticaRESUMEN
BACKGROUND: Apoptosis of vascular cells is considered to be a major determinant of atherosclerotic plaque vulnerability and potential rupture. Plasmin can be generated in atherosclerotic plaques and recent in vitro data suggest that plasminogen activation may trigger vascular smooth muscle cell (VSMC) apoptosis. AIM: To determine whether plasminogen activation may induce aortic VSMC apoptosis ex vivo and in vivo. METHODS AND RESULTS: Mice with single or combined deficiencies of apolipoprotein E (ApoE) and plasminogen activator inhibitor-1 (PAI-1) were used. Ex vivo incubation with plasminogen of isolated aortic tunica media from PAI-1-deficient mice induced plasminogen activation and VSMC apoptosis, which was inhibited by alpha2-antiplasmin. In vivo, levels of plasmin, active caspase 3 and VSMC apoptotic index were significantly higher in atherosclerotic aortas from mice with combined ApoE and PAI-1 deficiencies than in those from littermates with single ApoE deficiency. A parallel decrease in VSMC density was observed. CONCLUSIONS: These data strongly suggest that plasminogen activation may contribute to VSMC apoptosis in atherosclerotic plaques.
Asunto(s)
Aorta/metabolismo , Apoptosis , Aterosclerosis/metabolismo , Músculo Liso Vascular/metabolismo , Plasminógeno/metabolismo , Túnica Media/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/patología , Apolipoproteínas E/genética , Aterosclerosis/patología , Modelos Animales de Enfermedad , Fibrinolisina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Inhibidor 1 de Activador Plasminogénico/genética , Túnica Media/efectos de los fármacos , Túnica Media/patología , alfa 2-Antiplasmina/farmacologíaRESUMEN
Clinical studies reveal concomitant occurrence of several gastrointestinal and urologic disorders, including irritable bowel syndrome and interstitial cystitis. The purpose of this study was to determine the mechanisms underlying cross-organ sensitization at the level of dorsal root ganglion (DRG) after acute and subsided gastrointestinal inflammation. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and Fast Blue were injected into the distal colon and urinary bladder of male rats, respectively. Convergent DRG neurons were found in L1-L3 and L6-S2 ganglia with an average distribution of 14% +/- 2%. The resting membrane potential (RMP) of cells isolated from upper lumbar (UL) ganglia was -59.8 +/- 2.7 mV, whereas lumbosacral (LS) neurons were more depolarized (RMP = -49.4 +/- 2.1 mV, P < or = 0.05) under control conditions. Acute trinitrobenzene sulfonic acid (TNBS) colitis (3 days) decreased voltage and current thresholds for action potential firing in LS but not UL convergent capsaicin-sensitive neurons. This effect persisted for 30 days in the absence of overt colonic inflammation. The current threshold for action potential (AP) firing in UL cells was also decreased from 165.0 +/- 24.5 pA (control) to 85.0 +/- 19.1 pA at 30 days (P < or = 0.05), indicating increased excitability. The presence of a subpopulation of colon-bladder convergent DRG neurons and their persistent hyperexcitability after colonic inflammation provides a basis for pelvic organ cross-sensitization.
Asunto(s)
Colon/inervación , Colon/fisiopatología , Inflamación/fisiopatología , Vejiga Urinaria/inervación , Vejiga Urinaria/fisiopatología , Potenciales de Acción/fisiología , Animales , Cistitis Intersticial/complicaciones , Modelos Animales de Enfermedad , Ganglios Espinales/citología , Ganglios Espinales/fisiopatología , Inmunohistoquímica , Síndrome del Colon Irritable/complicaciones , Masculino , Neuronas/citología , Neuronas/fisiología , Ratas , Ratas Sprague-Dawley , Ácido Trinitrobencenosulfónico/efectos adversosAsunto(s)
Modelos Animales de Enfermedad , Factores de Crecimiento Endotelial/deficiencia , Linfocinas/deficiencia , Ratones Mutantes , Isquemia Miocárdica/patología , Miocardio/patología , Neovascularización Fisiológica/genética , Empalme Alternativo , Animales , Antígenos de Diferenciación , Factores de Crecimiento Endotelial/genética , Endotelio Vascular/química , Marcación de Gen , Pruebas de Función Cardíaca , Linfocinas/genética , Ratones , Músculo Esquelético/irrigación sanguínea , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Cancer patients treated for venous thromboembolism with low molecular weight heparin (LMWH) have a better survival rate than patients treated with unfractionated heparin (UFH). Because fibrin-associated angiogenesis is an important determinant in the progression and metastasis of many solid tumors, the effects of heparins on in vitro angiogenesis were investigated. Both UFH and LMWH inhibited bFGF-induced proliferation of human microvascular endothelial cells (hMVECs) to the same the extent (36-60%). VEGF165-induced proliferation was inhibited to a to a lesser extent (19-33%). Turbidity measurements and electron microscopy showed that the presence of LMWH during polymerization of the fibrin matrix led to a more transparent rigid network with thin fibrin bundles, whereas the presence of UFH resulted in a more opaque more porous network with thick fibrin fibers. We used a human in vitro angiogenesis model, which consisted of hMVECs seeded on top of a fibrin matrix, and stimulated the cells with basic fibroblast growth factor plus tumor necrosis factor a to induce capillary-like tubular structures. The formation of capillary-like tubular structures was retarded with matrices polymerized in the presence of LMWH (46% inhibition compared with a control matrix for both 1.5 and 10 units/ml LMWH), whereas matrices polymerized in the presence of UFH facilitated tubular structure formation (72 and 36% stimulation compared with a control matrix for 1.5 and 10 units/ml UFH, respectively). Similar results were obtained for cells stimulated with vascular endothelial growth factor plus tumor necrosis factor alpha. These data demonstrate the inhibitory effect of heparins on proliferation of hMVECs and provide a novel mechanism by which LMWH may affect tumor progression, namely reduced ingrowth of microvascular structures in a fibrinous stroma matrix by rendering it less permissive for invasion.
Asunto(s)
Fibrina/metabolismo , Fibrinolíticos/farmacología , Heparina de Bajo-Peso-Molecular/farmacología , Heparina/farmacología , Neovascularización Fisiológica/efectos de los fármacos , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/crecimiento & desarrollo , HumanosRESUMEN
Previous studies have shown that blocking endothelial protein C receptor (EPCR)-protein C interaction results in about an 88% decrease in circulating activated protein C (APC) levels generated in response to thrombin infusion and exacerbates the response to Escherichia coli. To determine whether higher levels of EPCR expression on endothelial cells might further enhance the activation of protein C and protect the host during septicemia, we generated a transgenic mouse (Tie2-EPCR) line which placed the expression of EPCR under the control of the Tie2 promoter. The mice express abundant EPCR on endothelial cells not only on large vessels, but also on capillaries where EPCR is generally low. Tie2-EPCR mice show higher levels of circulating APC after thrombin infusion. Upon infusion with factor Xa and phospholipids, Tie2-EPCR mice generate more APC, less thrombin and are protected from fibrin/ogen deposition compared with wild type controls. The Tie2-EPCR animals also generate more APC upon lipopolysaccharide (LPS) challenge and have a survival advantage. These results reveal that overexpression of EPCR can protect animals against thrombotic or septic challenge.
Asunto(s)
Factores de Coagulación Sanguínea/biosíntesis , Endotoxinas/metabolismo , Hemostasis , Receptor TIE-2/genética , Receptores de Superficie Celular/biosíntesis , Animales , Anticuerpos Monoclonales/química , Separación Celular , Progresión de la Enfermedad , Endotelio Vascular/citología , Escherichia coli/metabolismo , Fibrina/metabolismo , Fibrinógeno/metabolismo , Citometría de Flujo , Hemostáticos , Lipopolisacáridos/metabolismo , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Proteína C/metabolismo , Receptor TIE-2/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sepsis , Trombina/metabolismo , Trombosis , Factores de Tiempo , TransgenesRESUMEN
To investigate a potential role for stromelysin-1 (MMP-3) in the development and progression of atherosclerotic lesions and aneurysm formation, mice with a deficiency of apolipoprotein E (ApoE(-/-):MMP-3(+/+))) or with a combined deficiency of apoE and MMP-3 (ApoE(-/-):MMP-3(-/-)) were kept on a cholesterol-rich diet for 30 weeks. Atherosclerotic lesions throughout the thoracic aorta were significantly larger in ApoE(-/-):MMP-3(-/-) than in ApoE(-/-):MMP-3(+/+) mice (P<0.05) and contained more fibrillar collagen (P<0.01). Aneurysms in the thoracic and abdominal aortas were less frequent in ApoE(-/-):MMP-3(-/-) than in ApoE(-/-):MMP-3(+/+) mice (8.5+/-1.7% vs 14+/-2.1% of sections, mean+/-SD, P<0.01). Immunocytochemistry revealed enhanced accumulation of macrophages in atherosclerotic lesions of ApoE(-/-):MMP-3(+/+) mice (P<0.01) and expression of urokinase-type plasminogen activator (u-PA) and MMP-3 colocalizing with macrophages. Zymography confirmed the presence of u-PA and MMP-3 activity in extracts of atherosclerotic aortas. These data suggest that plasmin, generated by macrophage-secreted u-PA, activates pro-MMP-3 produced by accumulated macrophages. MMP-3 activity may then contribute to a reduction of plaque size, possibly by degradation of matrix components, and promote aneurysm formation by degradation of the elastica lamina.
Asunto(s)
Aneurisma/etiología , Arteriosclerosis/complicaciones , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/fisiología , Aneurisma/metabolismo , Aneurisma/patología , Animales , Antígenos de Diferenciación/análisis , Apolipoproteínas E/genética , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Dieta Aterogénica , Femenino , Genotipo , Inmunohistoquímica , Metabolismo de los Lípidos , Macrófagos/química , Masculino , Metaloproteinasa 3 de la Matriz/inmunología , Ratones , Ratones Noqueados , Activador de Plasminógeno de Tipo Uroquinasa/inmunología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Fluid flow modulates the synthesis and secretion by endothelial cells (ECs) of several proteins that control the hemostatic properties of the vessel wall. Tissue factor pathway inhibitor (TFPI), also synthesized by ECs, is the main downregulator of tissue factor-dependent procoagulant activity. In the present study, we investigated the effect of physiological shear stress on the expression, distribution, and release of TFPI in cultured ECs. The EA.hy926 cell line was grown in a hollow-fiber perfusion system and exposed for variable times to different shear values: 0.27 dyne/cm(2) (minimal flow), 4.1 dyne/cm(2) (venous flow), and 19 dyne/cm(2) (moderate arterial flow). Step increase of the shear stress from 0.27 to 19 dyne/cm(2) induced a sharp increase of TFPI released into the medium and a parallel decrease and redistribution of cell-associated TFPI, which suggests that an acute release of TFPI occurred from the cellular pools. During 24 hours of high shear stress, cell-associated TFPI antigen and mRNA increased time-dependently. Subjecting ECs to steady shear stress for 72 hours also upregulated the expression and production of TFPI, in direct correlation with the degree of the shear. The secretion of TFPI was enhanced 1.9-fold under venous flow and 2.4-fold under arterial flow compared with minimal flow. Equally, cell-associated TFPI antigen and cell surface TFPI activity increased proportionally with the shear stress. The expression of TFPI mRNA, as determined by Northern blotting, increased up to 2-fold in ECs under venous flow and up to 3-fold under arterial flow. These results suggest that shear forces regulate TFPI by modulating its release and gene expression in ECs in vitro.
Asunto(s)
Lipoproteínas/biosíntesis , Lipoproteínas/metabolismo , Regulación hacia Arriba/fisiología , Velocidad del Flujo Sanguíneo , Capilares/química , Capilares/fisiología , Capilares/ultraestructura , Resistencia Capilar/fisiología , Línea Celular Transformada , Endotelio Vascular/química , Endotelio Vascular/citología , Endotelio Vascular/crecimiento & desarrollo , Inhibidores del Factor Xa , Humanos , Lipoproteínas/análisis , Lipoproteínas/sangre , Flujo Sanguíneo Regional/fisiología , Estrés MecánicoRESUMEN
Angiogenesis and activated blood coagulation are involved in tumor growth and metastasis. Although some have suggested that activation of coagulation in tumors is not linked to activation of platelets, no data exist to either support or refute this concept. However, platelet turnover in cancer patients is often increased, and platelets are carriers of angiogenic growth factors. We hypothesized that platelets are involved in tumor-associated angiogenesis. To obtain evidence supporting this hypothesis, we have studied whether the angiogenic and coagulation pathways and platelets are concomitantly activated in cancer patients with soft tissue sarcomas (STSs) using a novel method to detect activated platelets in tumor specimens. Twelve patients with STS were selected on the basis of having intratumoral accumulation of fluid, which was aspirated. These accumulations demonstrated very high concentrations of vascular endothelial growth factor and coagulation factors (including thrombin-antithrombin-complex). Tumor specimens showed dense vascularization with intense vascular endothelial growth factor expression and the presence of activated platelets. Taken together, these results support the concept that angiogenesis, blood coagulation, and platelets are concomitantly activated in STS and support the hypothesis that platelets contribute to tumor-induced angiogenesis.
Asunto(s)
Plaquetas/patología , Factores de Crecimiento Endotelial/análisis , Linfocinas/análisis , Neovascularización Patológica , Activación Plaquetaria , Sarcoma/patología , Sarcoma/fisiopatología , Antitrombina III/análisis , Histiocitoma Fibroso Benigno/irrigación sanguínea , Histiocitoma Fibroso Benigno/patología , Humanos , Leiomiosarcoma/irrigación sanguínea , Leiomiosarcoma/patología , Péptido Hidrolasas/análisis , Sarcoma/irrigación sanguínea , Trombomodulina/análisis , Tromboplastina/análisis , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Both sexes of adult mice homozygous for a targeted mutation of the Igf1 gene, encoding insulin-like growth factor 1, are infertile dwarfs (approximately 30% of normal size). The testes are reduced in size less than expected from the degree of dwarfism but sustain spermatogenesis only at 18% of the normal level. The epididymides are overall nearly allometric to the reduced body weight, but the distal regions of the duct, vas deferens, seminal vesicles, and prostate are vestigial. Despite the mutational impact on the epididymis, capacitated sperm are able to fertilize wild type eggs in vitro. It is hypothesized that the infertility of male mutants is caused by failure of androgenization resulting in absence of mating behavior, due to drastically reduced levels of serum testosterone (18% of normal). This hormonal deficiency was correlated with an ultrastructural analysis of mutant Leydig cells revealing a significant developmental delay, while assays in organ culture showed that the basal and LH-stimulated production of testosterone by testicular parenchyma is reduced in comparison with wild type controls. The female mutants fail to ovulate even after administration of gonadotropins, which is apparently the primary cause of their infertility, and possess an infantile uterus that exhibits a dramatic hypoplasia especially in the myometrium. The phenotypic manifestations of the mutation were correlated with the localization of transcripts for insulin-like growth factor I and its cognate receptor in wild type reproductive tissues by in situ hybridization.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/genética , Mutación , Reproducción/genética , Animales , Epidídimo/metabolismo , Epidídimo/patología , Femenino , Regulación del Desarrollo de la Expresión Génica , Infertilidad/genética , Factor I del Crecimiento Similar a la Insulina/fisiología , Masculino , Ratones , Ratones Mutantes , Tamaño de los Órganos , Ovario/metabolismo , Ovario/patología , Motilidad Espermática , Testículo/metabolismo , Testículo/patología , Testosterona/sangre , Útero/metabolismoRESUMEN
Free radicals contribute to the pathogenesis of diabetic cardiomyopathy. We present a method for in vivo observation of free radical events within murine diabetic cardiomyopathy. This study reports on in vivo imaging of protein/lipid radicals using molecular MRI (mMRI) and immuno-spin trapping (IST) in diabetic cardiac muscle. To detect free radicals in diabetic cardiomyopathy, streptozotocin (STZ)-exposed mice were given 5,5-dimethyl-pyrroline-N-oxide (DMPO) and administered an anti-DMPO probe (biotin-anti-DMPO antibody-albumin-Gd-DTPA). For controls, non-diabetic mice were given DMPO (non-disease control), and administered an anti-DMPO probe; or diabetic mice were given DMPO but administered a non-specific IgG contrast agent instead of the anti-DMPO probe. DMPO administration started at 7 weeks following STZ treatment for 5 days, and the anti-DMPO probe was administered at 8 weeks for MRI detection. MRI was used to detect a significant increase (p < 0.001) in MRI signal intensity (SI) from anti-DMPO nitrone adducts in diabetic murine left-ventricular (LV) cardiac tissue, compared to controls. Regional increases in MR SI in the LV were found in the apical and upper-left areas (p < 0.01 for both), compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised cardiac tissues, which indicated elevated fluorescence only in cardiac muscle of mice administered the anti-DMPO probe. Oxidized lipids and proteins were also found to be significantly elevated (p < 0.05 for both) in diabetic cardiac muscle compared to controls. It can be concluded that diabetic mice have more heterogeneously distributed radicals in cardiac tissue than non-diabetic mice.
Asunto(s)
Cardiomiopatías Diabéticas/patología , Imagen por Resonancia Magnética , Detección de Spin , Albúminas/química , Animales , Medios de Contraste/química , Óxidos N-Cíclicos/química , Diabetes Mellitus Experimental/patología , Radicales Libres/química , Gadolinio DTPA/química , Ventrículos Cardíacos/patología , Lípidos/química , Ratones , Ratones Endogámicos C57BL , Modelos Químicos , Miocitos Cardíacos/metabolismo , Estrés Oxidativo , Oxígeno/química , EstreptozocinaRESUMEN
The appearance and accumulation of apolipoprotein B and unesterified cholesterol in the lesion-prone areas of the aorta in rabbits with diet-induced hyperlipidemia were investigated by histo-, and cytochemical techniques. Apolipoprotein B was detected by an indirect immunoperoxidase procedure both in the light and electron microscopy. Unesterified cholesterol was revealed using filipin and tomatine as specific probes. In the prelesional stages of atherogenesis, before the appearance of any structurally detectable lesions, as demonstrated by bright-field and fluorescence microscopy, apolipoprotein B and free cholesterol accumulated progressively in the extracellular matrix of the subendothelial space. At ultrastructural level, extracellular phospholipid liposomes, unesterified cholesterol and apolipoprotein B concomitantly appeared and accumulated focally in the same areas. Apolipoprotein B was preferentially located on the outer surface of the free cholesterol-containing phospholipid lamellae of the extracellular liposomes. In the lesional stages leading to fatty streak formation, the extracellular liposomes, apolipoprotein B and unesterified cholesterol had also topographically a superimposed localization pattern. Intracellular apolipoprotein B and unesterified cholesterol were also colocalized in some intimal lipid-laden cells. In the prelesional stages of hyperlipidemia the prevalent localization of apolipoprotein B around individual unesterified cholesterol-rich extracellular phospholipid liposomes, progressively accumulating in the subendothelial space, suggests their possible origin from serum-derived lipoproteins.