RESUMO
Humans carrying the factor V Leiden (FVL) variant have a fivefold increased risk for venous thrombosis. However, incidence of deep vein thrombosis (DVT) is proportionally greater than that of pulmonary embolism (PE) in these individuals. This is known as the FVL paradox. We hypothesized that the rate of initial DVT development is similar in FVL and noncarriers, but thrombi in FVL carriers are more stable and develop into a clinically significant DVT more often than in noncarriers. To test this, we induced thrombi in the femoral vein of wild-type (WT), heterozygous (F5L/+), and FVL homozygous (F5L/L) mice. Using intravital microscopy, thrombus size and embolization were visualized and emboli in the lungs were quantified. Compared with WT, femoral vein thrombi in F5L/+ and F5L/L mice were larger and embolized less. Total and large embolic events, the percentage of thrombus that embolized, and PE burden were significantly decreased in F5L/L mice. This suggests that in noncarriers (reflected by WT), a minor injury initially resulting in a small DVT tends to remain small and asymptomatic because of the embolization of the otherwise growing thrombus. Alternatively, the same insult in people with FVL (reflected by F5L/L) leads to thrombus growth as a result of less embolization, and thus symptomatic DVT development.
Assuntos
Coagulação Sanguínea/genética , Fator V/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Trombose Venosa/diagnóstico , Trombose Venosa/genética , Animais , Biomarcadores , Biópsia , Testes de Coagulação Sanguínea , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Trombose Venosa/sangueRESUMO
Most inherited hemostatic disorders exhibit incomplete penetrance and variable expressivity, which can be because of genetic or environmental interactions. This wide phenotypic variability for a given disease can be partly explained by modifier gene interactions. Modifier gene interactions have been described for VWD, TTP and venous thrombosis associated with the factor V Leiden mutation. We have exploited advances in mouse genetics in an effort to identify novel genetic loci that may serve as candidate genetic modifiers for bleeding and thrombosis in humans. We have identified several loci affecting plasma VWF levels and have identified and characterized mouse models of ADAMTS13 deficiency and Factor V Leiden that could be useful for identifying novel genes contributing to thrombosis risk in humans.
Assuntos
Hemostasia/genética , Trombose/genética , Animais , Transtornos da Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/genética , Genes , HumanosRESUMO
Elevated expression of plasminogen activator inhibitor-1 (PAI-1) in tumors is associated with a poor prognosis in many cancers. Reduced tumor growth and angiogenesis have also been reported in mice deficient in PAI-1. These results suggest that PAI-1 may be required for efficient angiogenesis and tumor growth. In the present study, we demonstrate that PAI-1 can both enhance and inhibit the growth of M21 human melanoma tumors in nude mice and that this appears to be due to PAI-1 regulation of angiogenesis. Quantitative analysis of angiogenesis in a Matrigel implant assay indicated that in PAI-1 null mice angiogenesis was reduced approximately 60% compared with wild-type mice, while in mice overexpressing PAI-1, angiogenesis was increased nearly 3-fold. Furthermore, addition of PAI-1 to implants in wild-type mice enhanced angiogenesis up to 3-fold at low concentrations but inhibited angiogenesis nearly completely at high concentrations. Together, these data demonstrate that PAI-1 is a potent regulator of angiogenesis and hence of tumor growth and suggest that understanding the mechanism of this activity may lead to the development of important new therapeutic agents for controlling pathologic angiogenesis.
Assuntos
Melanoma/patologia , Neovascularização Patológica , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Animais , Divisão Celular , Colágeno/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Humanos , Imuno-Histoquímica , Laminina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Transplante de Neoplasias , Prognóstico , Proteoglicanas/metabolismo , Células Tumorais Cultivadas , Vitronectina/metabolismo , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Tissue factor initiates blood coagulation after atherosclerotic plaque disruption. Tissue factor pathway inhibitor (TFPI) inhibits tissue factor activity and may reduce thrombus formation in this setting. We evaluated the effect of heterozygous TFPI deficiency on the development of atherosclerosis and thrombosis in atherosclerosis-prone mice. METHODS AND RESULTS: Mice with a combined heterozygous TFPI deficiency and homozygous apolipoprotein E deficiency (TFPI(+/-)/apoE(-/-)) were generated by crossbreeding, and they were analyzed for atherosclerosis throughout the vascular tree. Compared with mice with a normal TFPI genotype (TFPI(+/+)/apoE(-/-)), mice with a TFPI deficiency exhibited a greater atherosclerotic burden involving the carotid and common iliac arteries. Staining for active tissue factor within the plaque revealed more activity in TFPI(+/-)/apoE(-/-) mice compared with TFPI(+/+)/apoE(-/-) mice. Consistent with increased plaque tissue factor activity, the time to occlusive thrombosis after photochemical carotid plaque injury was significantly decreased in TFPI(+/-)/apoE(-/-) mice. CONCLUSIONS: These observations indicate that TFPI protects from atherosclerosis and is an important regulator of the thrombosis that occurs in the setting of atherosclerosis.
Assuntos
Arteriosclerose/metabolismo , Lipoproteínas/deficiência , Trombose/metabolismo , Animais , Apolipoproteínas E/genética , Arteriosclerose/etiologia , Arteriosclerose/patologia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/complicações , Fator VIIa/metabolismo , Feminino , Genótipo , Lipoproteínas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tromboplastina , Trombose/etiologia , Trombose/patologiaRESUMO
Increased fibrin deposition following inflammatory lung injury has been proposed to facilitate the development of pulmonary fibrosis. Therefore, factors predisposing to thrombosis may affect the fibrotic response to injury. Activated protein C (aPC) resistance due to the factor V Leiden mutation (FvL) is a common genetic risk factor for vascular thrombosis. To examine the relationship between aPC resistance and the development of pulmonary fibrosis, lung inflammation was induced by bleomycin in mice carrying the FvL mutation. Three weeks following the instillation of 0.0375 U of bleomycin, the lungs of mice homozygous and heterozygous for FvL contained significantly more hydroxyproline (35 +/- 4 and 36 +/- 7 ug hydroxyproline/mg total protein, respectively) than wild-type mice (26 +/- 6 ug/mg protein, p <0.01 for both comparisons). These data demonstrate a strong relationship between aPC resistance and the pulmonary fibrosis that occurs following inflammatory lung injury.
Assuntos
Fator V/genética , Fibrose Pulmonar/etiologia , Resistência à Proteína C Ativada/complicações , Animais , Bleomicina , Modelos Animais de Doenças , Hidroxiprolina/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pneumonia/induzido quimicamente , Mutação PuntualRESUMO
Dissolution of the fibrin blood clot is regulated in large part by plasminogen activator inhibitor-1 (PAI-1). Elevated levels of plasma PAI-1 may be an important risk factor for atherosclerotic vascular disease and are associated with premature myocardial infarction. The role of the endogenous plasminogen activation system in limiting thrombus formation following atherosclerotic plaque disruption is unknown. This study found that genetic deficiency for PAI-1, the primary physiologic regulator of tissue-type plasminogen activator (tPA), prolonged the time to occlusive thrombosis following photochemical injury to carotid atherosclerotic plaque in apolipoprotein E-deficient (apoE(-/-)) mice. However, anatomic analysis revealed a striking difference in the extent of atherosclerosis at the carotid artery bifurcation between apoE(-/-) mice and mice doubly deficient for apoE and PAI-1 (PAI-1(-/-)/apoE(-/-)). Consistent with a previous report, PAI-1(+/+)/apoE(-/-)and PAI-1(-/-)/apoE(-/-) mice developed similar atherosclerosis in the aortic arch. The marked protection from atherosclerosis progression at the carotid bifurcation conferred by PAI-1 deficiency suggests a critical role for PAI-1 in the pathogenesis of atherosclerosis at sites of turbulent flow, potentially through the inhibition of fibrin clearance. Consistent with this hypothesis, intense fibrinogen/fibrin staining was observed in atherosclerotic lesions at the carotid bifurcation compared to the aortic arch. These observations identify significant differences in the pathogenesis of atherosclerosis at varying sites in the vascular tree and suggest a previously unappreciated role for the plasminogen activation system in atherosclerosis progression at sites of turbulent flow. (Blood. 2000;96:4212-4215)
Assuntos
Aorta Torácica/patologia , Doenças da Aorta/prevenção & controle , Arteriosclerose/prevenção & controle , Doenças das Artérias Carótidas/prevenção & controle , Artéria Carótida Primitiva/patologia , Estenose das Carótidas/prevenção & controle , Inibidor 1 de Ativador de Plasminogênio/deficiência , Plasminogênio/fisiologia , Animais , Aorta Torácica/química , Doenças da Aorta/genética , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Arteriosclerose/genética , Arteriosclerose/patologia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Lesões das Artérias Carótidas , Artéria Carótida Primitiva/química , Estenose das Carótidas/genética , Estenose das Carótidas/patologia , Progressão da Doença , Fibrina/análise , Fibrinogênio/análise , Hemorreologia , Masculino , Camundongos , Camundongos Knockout , Fotoquímica , Inibidor 1 de Ativador de Plasminogênio/genéticaRESUMO
A polymorphism in coagulation factor V, factor V Leiden (FVL), is the major known genetic risk factor for thrombosis in humans. Approximately 10% of mutation carriers experience clinically significant thrombosis in their lifetime. In a small subset of patients, thrombosis is associated with coinheritance of other prothrombotic gene mutations. However, the potential contribution of additional genetic risk factors in the majority of patients remains unknown. To gain insight into the molecular basis for the variable expressivity of FVL, mice were generated carrying the homologous mutation (R504Q [single-letter amino acid codes]) inserted into the endogenous murine Fv gene. Adult heterozygous (FvQ/+) and homozygous (FvQ/Q) mice are viable and fertile and exhibit normal survival. Compared with wild-type mice, adult FvQ/Q mice demonstrate a marked increase in spontaneous tissue fibrin deposition. No differences in fetal development or survival are observed among FvQ/Q, FvQ/+ or control littermates on the C57BL/6J genetic background. In contrast, on a mixed 129Sv-C57BL/6J genetic background, FvQ/Q mice develop disseminated intravascular thrombosis in the perinatal period, resulting in significant mortality shortly after birth. These results may explain the high degree of conservation of the R504/R506 activated protein C cleavage site within FV among mammalian species and suggest an important contribution of other genetic factors to the thrombosis associated with FVL in humans. (Blood. 2000;96:4222-4226)
Assuntos
Resistência à Proteína C Ativada/genética , Modelos Animais de Doenças , Fator V/genética , Trombose/etiologia , Substituição de Aminoácidos , Animais , Animais Recém-Nascidos , Cruzamentos Genéticos , Coagulação Intravascular Disseminada/genética , Epistasia Genética , Fator V/fisiologia , Feminino , Fertilidade , Fibrina/metabolismo , Marcação de Genes , Genes Letais , Longevidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Fenótipo , Mutação Puntual , Splicing de RNA , Fatores de RiscoRESUMO
The increased risk of hyperlipidemia on the development of complications of atherosclerosis is well established. Cholesterol-lowering therapies lead to a decrease in the incidence of vascular thrombotic events that is out of proportion to the reduction in plaque size. This suggests that the occurrence of acute thrombosis overlying a disrupted plaque is influenced by changes in lipid levels. The influence of acute hyperlipidemia on the development of thrombosis overlying an atherosclerotic plaque in vivo has not been extensively studied. We used a murine model of vascular injury induced by a photochemical reaction to elicit thrombus formation overlying an atherosclerotic plaque. Fifteen apolipoprotein E-deficient mice were maintained on normal chow until the age of 30 weeks. Five days before the induction of thrombosis, 6 mice were started on a high fat diet, and 9 mice were continued on normal chow. Mice then underwent photochemical injury to the common carotid artery immediately proximal to the carotid bifurcation, where an atherosclerotic plaque is consistently present. Mice maintained on normal chow developed occlusive thrombi, determined by cessation of blood flow, 44+/-5 minutes (mean+/-SEM) after photochemical injury, whereas mice fed a high fat chow developed occlusive thrombosis at 27+/-3 minutes (P<0.02). Histological analysis confirmed the presence of acute thrombus formation overlying an atherosclerotic plaque. These studies demonstrate a useful model for assessing the determinants of thrombosis in the setting of atherosclerosis and show that acute elevations in plasma cholesterol facilitate thrombus formation at sites of atherosclerosis after vascular injury.
Assuntos
Apolipoproteínas E/genética , Doenças das Artérias Carótidas/complicações , Lesões das Artérias Carótidas/complicações , Hiperlipidemias/complicações , Trombose/etiologia , Animais , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Colesterol/sangue , Dieta Aterogênica , Gorduras na Dieta/administração & dosagem , Modelos Animais de Doenças , Feminino , Corantes Fluorescentes , Hiperlipidemias/genética , Hiperlipidemias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Rosa Bengala , Trombose/genética , Trombose/patologiaRESUMO
Occlusive thrombosis depends on the net balance between platelets, coagulation, and fibrinolytic factors. Epidemiologic information suggests that plasminogen activator inhibitor-1 (PAI-1), a central regulator of the fibrinolytic system, plays an important role in determining the overall risk for clinically significant vascular thrombosis. Vitronectin (VN), an abundant plasma and matrix glycoprotein, binds PAI-1 and stabilizes its active conformation. This study assessed the role of PAI-1 and VN expression in the formation of occlusive vascular thrombosis following arterial or venous injury. The common carotid arteries of 17 wild-type (WT) mice and 8 mice deficient in PAI-1 were injured photochemically while blood flow was continuously monitored. WT mice developed occlusive thrombi at 52.0 +/- 3.8 minutes (mean +/- SEM) following injury; mice deficient in PAI-1 developed occlusive thrombosis at 127 +/- 15 minutes (P <.0001). Mice deficient in VN (n = 12) developed vascular occlusion 77 +/- 11 minutes after injury, intermediate between the values observed for WT mice (P <.03) and mice deficient in PAI-1 (P <.01). PAI-1 and VN also affected the time to occlusion after injury to the jugular vein. Three WT mice developed occlusive venous thrombosis an average of 39.7 +/- 1 minutes following the onset of injury, whereas the jugular veins of 4 mice deficient in PAI-1 and 4 deficient in VN occluded 56.7 +/- 5 and 58.7 +/- 2 minutes, respectively, following injury (P <.04 and P <.01 compared to WT mice). These results suggest that endogenous fibrinolysis and its regulation by PAI-1 and VN have important roles in the development of occlusive vascular thrombosis after vascular injury. (Blood. 2000;95:577-580)
Assuntos
Estenose das Carótidas/fisiopatologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Trombose/fisiopatologia , Trombose Venosa/fisiopatologia , Vitronectina/fisiologia , Animais , Estenose das Carótidas/sangue , Estenose das Carótidas/genética , Veias Jugulares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidor 1 de Ativador de Plasminogênio/deficiência , Inibidor 1 de Ativador de Plasminogênio/genética , Trombose/sangue , Trombose/genética , Trombose Venosa/sangue , Trombose Venosa/genética , Vitronectina/deficiência , Vitronectina/genéticaRESUMO
We have identified altered lineage-specific expression of an N-acetylgalactosaminyltransferase gene, Galgt2, as the gain-of-function mechanism responsible for the action of the Mvwf locus, a major modifier of plasma von Willebrand factor (VWF) level in RIIIS/J mice. A switch of Galgt2 gene expression from intestinal epithelial cell-specific to a pattern restricted to the vascular endothelial cell bed leads to aberrant posttranslational modification and rapid clearance of VWF from plasma. Transgenic expression of Galgt2 directed to vascular endothelial cells reproduces the low VWF phenotype, confirming this switch in lineage-specific gene expression as the likely molecular mechanism for Mvwf. These findings identify alterations in glycosyltransferase function as a potential general mechanism for the genetic modification of plasma protein levels.
Assuntos
N-Acetilgalactosaminiltransferases/metabolismo , Fator de von Willebrand/metabolismo , Animais , Linhagem Celular , Linhagem da Célula , Endotélio Vascular/citologia , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , N-Acetilgalactosaminiltransferases/genética , Polimorfismo Genético , Fator de von Willebrand/genéticaRESUMO
Plasminogen activator inhibitor-2 (PAI-2), a member of the serpin gene family, is thought to serve as a primary regulator of plasminogen activation in the extravascular compartment. High levels of PAI-2 are found in keratinocytes, monocytes, and the human trophoblast, the latter suggesting a role in placental maintenance or embryo development. The primarily intracellular distribution of PAI-2 also may indicate a unique regulatory role in a protease-dependent cellular process such as apoptosis. To examine the potential functions of PAI-2 in vivo, we generated PAI-2-deficient mice by gene targeting in embryonic stem cells. Homozygous PAI-2-deficient mice exhibited normal development, survival, and fertility and were also indistinguishable from normal controls in response to a bacterial infectious challenge or endotoxin infusion. No differences in monocyte recruitment into the peritoneum were observed after thioglycollate injection. Epidermal wound healing was equivalent among PAI-2 -/- null and control mice. Finally, crossing PAI-2 -/- with PAI-1 -/- mice to generate animals deficient in both plasminogen activator inhibitors failed to uncover an overlap in function between these two related proteins.
Assuntos
Inibidor 2 de Ativador de Plasminogênio/genética , Animais , Southern Blotting , Marcação de Genes/métodos , Genótipo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Monócitos/imunologia , Fenótipo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Células-Tronco/metabolismo , Cicatrização/genéticaRESUMO
Type 1 von Willebrand disease (VWD) is a common inherited disorder characterized by mild to moderate bleeding and reduced levels of von Willebrand factor (VWF). An animal model for human type 1 VWD, the RIIIS/J mouse strain, exhibits a prolonged bleeding time and reduced plasma VWF levels. We have previously mapped the defect in RIIIS/J to distal mouse Chr 11, distinct from the Vwf locus on Chr 6. This locus, Mvwf, was localized to an approximately 0.5-cM interval, tightly linked to Gip, distal to Ngfr, and proximal to Hoxb. We have now used these genetic markers to construct a contig of yeast and bacterial artificial chromosomes and bacteriophage P1 clones spanning the approximately 300-kb Mvwf nonrecombinant interval. In a comparative mapping approach, mouse homologues of mapped human expressed sequence tags (ESTs) were localized relative to the candidate interval. Twenty-one sequence-tagged sites and ESTs from the corresponding human syntenic region 17q21.3 were ordered using the high-resolution Stanford TNG3 radiation hybrid panel. Based on the resulting radiation hybrid map and our mouse genetic and physical maps, the order of human and mouse genes in a >0.7-cM region appears to be conserved. Six genes localized to the Mvwf nonrecombinant interval by comparative mapping included orthologs of GNGT2, ATP6N1, and a nuclear domain protein. Seven other genes or ESTs were excluded from the candidate interval, including orthologs of PHB, PDK2, a speckle-type protein, and a UDP-galactose transporter. Using exon trapping, 10 additional putative expressed sequences were identified within the Mvwf nonrecombinant interval, including a previously cloned murine glycosyltransferase as well as exons showing sequence similarity to genes for Caenorhabditis elegans and Saccharomyces cerevisiae predicted proteins, an Arabidopsis thaliana ubiquitin-conjugating enzyme, and a Gallus gallus mRNA zipcode-binding protein. Further characterization of these putative genes could identify the dominant mutation responsible for low plasma VWF levels in RIIIS/J mice. These data may also aid in the localization of other disease loci mapped to this region, including the gene for tricho-dento-osseous syndrome and a murine locus for susceptibility to ozone-induced acute lung injury.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 17/genética , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Animais , Cromossomos/genética , Modelos Animais de Doenças , Éxons/genética , Etiquetas de Sequências Expressas , Marcadores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Reação em Cadeia da Polimerase/métodos , Proibitinas , RNA/análise , Mapeamento por Restrição , Sitios de Sequências Rotuladas , Doenças de von Willebrand/sangueRESUMO
Type 1 von Willebrand disease (VWD), characterized by reduced levels of plasma von Willebrand factor (VWF), is the most common inherited bleeding disorder in humans. Penetrance of VWD is incomplete, and expression of the bleeding phenotype is highly variable. In addition, plasma VWF levels vary widely among normal individuals. To identify genes that influence VWF level, we analyzed a genetic cross between RIIIS/J and CASA/Rk, two strains of mice that exhibit a 20-fold difference in plasma VWF level. DNA samples from F2 progeny demonstrating either extremely high or extremely low plasma VWF levels were pooled and genotyped for 41 markers spanning the autosomal genome. A novel locus accounting for 63% of the total variance in VWF level was mapped to distal mouse chromosome 11, which is distinct from the murine Vwf locus on chromosome 6. We designated this locus Mvwf for "modifier of VWF." Additional genotyping of as many as 2407 meioses established a high resolution genetic map with gene order Cola1-Itg3a-Ngfr-Mvwf/Gip-Hoxb9-Hoxb1++ +-Cbx'rs2-Cox5a-Gfap. The Mvwf candidate interval between Ngfr and Hoxb9 is approximately 0.5 centimorgan (cM). These results demonstrate that a single dominant gene accounts for the low VWF phenotype of RIIIS/J mice in crosses with several other strains. The pattern of inheritance suggests a gain-of-function mutation in a unique component of VWF biosynthesis or processing. Characterization of the human homologue for Mvwf may have relevance for a subset of type 1 VWD cases and may define an important genetic factor modifying penetrance and expression of mutations at the VWF locus.