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2.
J Thromb Haemost ; 16(4): 734-748, 2018 04.
Article in English | MEDLINE | ID: mdl-29418058

ABSTRACT

Essentials Tissue factor (TF) enhances factor VIIa (FVIIa) activity through structural and dynamic changes. We analyzed conservation of TF-activated FVIIa allosteric networks in extant vertebrate lamprey. Lamprey Tf/FVIIa molecular dynamics show conserved Tf-induced structural/dynamic FVIIa changes. Lamprey Tf activation of FVIIa allosteric networks follows molecular pathways similar to human. SUMMARY: Background Previous studies have provided insight into the molecular basis of human tissue factor (TF) activation of activated factor VII (FVIIa). TF-induced allosteric networks of FVIIa activation have been rationalized through analysis of the dynamic changes and residue connectivities in the human soluble TF (sTF)/FVIIa complex structure during molecular dynamics (MD) simulation. Evolutionary conservation of the molecular mechanisms for TF-induced allosteric FVIIa activation between humans and extant vertebrate jawless fish (lampreys), where blood coagulation emerged more than 500 million years ago, is unknown and of considerable interest. Objective To model the sTf/FVIIa complex from cloned Petromyzon marinus lamprey sequences, and with comparisons to human sTF/FVlla investigate conservation of allosteric mechanisms of FVIIa activity enhancement by soluble TF using MD simulations. Methods Full-length cDNAs of lamprey tf and f7 were cloned and characterized. Comparative models of lamprey sTf/FVIIa complex and free FVIIa were determined based on constructed human sTF/FVIIa complex and free FVIIa models, used in full-atomic MD simulations, and characterized using dynamic network analysis approaches. Results Allosteric paths of correlated motion from Tf contact points in lamprey sTf/FVIIa to the FVIIa active site were determined and quantified, and were found to encompass residue-residue interactions along significantly similar paths compared with human. Conclusions Despite low conservation of residues between lamprey and human proteins, 30% TF and 39% FVII, the structural and protein dynamic effects of TF activation of FVIIa appear conserved and, moreover, present in extant vertebrate proteins from 500 million years ago when TF/FVIIa-initiated extrinsic pathway blood coagulation emerged.


Subject(s)
Blood Coagulation , Evolution, Molecular , Factor VIIa/metabolism , Fish Proteins/metabolism , Lampreys/metabolism , Thromboplastin/metabolism , Allosteric Regulation , Amino Acid Sequence , Animals , Cloning, Molecular , Conserved Sequence , Factor VIIa/chemistry , Factor VIIa/genetics , Fish Proteins/chemistry , Fish Proteins/genetics , Humans , Lampreys/blood , Lampreys/genetics , Molecular Dynamics Simulation , Protein Conformation , Prothrombin Time , Structure-Activity Relationship , Thromboplastin/chemistry , Thromboplastin/genetics
3.
Hamostaseologie ; 35(1): 11-6, 2015.
Article in English | MEDLINE | ID: mdl-25666572

ABSTRACT

The endothelium is a widely distributed organ system that plays an important role in health and disease. The endothelium is remarkably heterogeneous in structure and function. One vital function of the endothelium is to maintain blood in its fluid state, and to provide controlled haemostasis at sites of vascular injury. In keeping with the theme of endothelial cell heterogeneity, endothelial cells from different sites of the vascular employ different strategies to mediate local haemostatic balance. These differences are sufficient to explain why systemic imbalances of haemostatic components invariably lead to local thrombotic phenotypes. An important goal for the future is to identify diagnostic markers that reflect phenotypic changes at the level of individual vascular beds, and to develop therapies that target one or another site of the vasculature.


Subject(s)
Blood Coagulation Factors/metabolism , Blood Coagulation , Blood Vessels/physiopathology , Endothelium, Vascular/physiopathology , Hemostasis , Thrombosis/physiopathology , Animals , Humans , Models, Cardiovascular
4.
J Thromb Haemost ; 11 Suppl 1: 46-66, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23809110

ABSTRACT

Every biological trait requires both a proximate and evolutionary explanation. The field of vascular biology is focused primarily on proximate mechanisms in health and disease. Comparatively little attention has been given to the evolutionary basis of the cardiovascular system. Here, we employ a comparative approach to review the phylogenetic history of the blood vascular system and endothelium. In addition to drawing on the published literature, we provide primary ultrastructural data related to the lobster, earthworm, amphioxus, and hagfish. Existing evidence suggests that the blood vascular system first appeared in an ancestor of the triploblasts over 600 million years ago, as a means to overcome the time-distance constraints of diffusion. The endothelium evolved in an ancestral vertebrate some 540-510 million years ago to optimize flow dynamics and barrier function, and/or to localize immune and coagulation functions. Finally, we emphasize that endothelial heterogeneity evolved as a core feature of the endothelium from the outset, reflecting its role in meeting the diverse needs of body tissues.


Subject(s)
Biological Evolution , Blood Vessels/growth & development , Endothelium, Vascular/growth & development , Animals , Humans , Phylogeny
5.
J Thromb Haemost ; 11(9): 1742-50, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23848403

ABSTRACT

BACKGROUND: Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt-related transcription factor 1 (RUNX1) of FPD patients. OBJECTIVES: To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1-mediated induction of the FPD phenotype. METHODS: We assessed whether RUNX1 and its mutants, in combination with E26 transformation-specific-1 (ETS-1), Core-binding factor subunit beta (CBFß), and Friend leukemia virus integration 1 (FLI-1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real-time RT-PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos-7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos-7 cells. RESULTS: We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS-1 and CBFß, or FLI-1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA-binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS-1, CBFß, and FLI-1. CONCLUSIONS: RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.


Subject(s)
Blood Platelet Disorders/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Gene Expression Regulation/genetics , Mutation , Platelet Factor 4/genetics , Proto-Oncogene Proteins c-ets/metabolism , Cell Line , Core Binding Factor Alpha 2 Subunit/metabolism , Electrophoretic Mobility Shift Assay , Humans , Real-Time Polymerase Chain Reaction , Subcellular Fractions/metabolism
6.
J Thromb Haemost ; 9 Suppl 1: 118-29, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21781247

ABSTRACT

The goal of this review is to examine the events that led to discovery of blood circulation. The Ancient Greeks, including Hippocrates and Galen viewed the cardiovascular system as comprising two distinct networks of arteries and veins. Galen claimed that the liver produced blood that was then distributed to the body in a centrifugal manner, whereas air or pneuma was absorbed from the lung into the pulmonary veins and carried by arteries to the various tissues of the body. Arteries also contained blood, which passed from the venous side via invisible pores in the interventricular septum and peripheral anastomoses. This was an open-ended system in which blood and air simply dissipated at the ends of veins and arteries according to the needs of the local tissue. Blood was not seen to circulate but rather to slowly ebb and flow. This view would hold sway for 15 centuries until 1628 when William Harvey published his momentous 72-page book, On the Motion of the Heart and Blood in Animals. Harvey employed experiment and deductive logic to show that arteries and veins are functionally, if not structurally, connected in the lung and the peripheral tissues, and that blood circulates. The mechanical force of the heart replaced Galen's elusive attractive powers. Ultimately, Galenism would collapse under the weight of Harvey's evidence, and a new paradigm of blood circulation would prevail.


Subject(s)
Blood Circulation , Cardiovascular System , History, 16th Century , History, 17th Century , History, Ancient , Humans
7.
J Thromb Haemost ; 7(8): 1384-92, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19496923

ABSTRACT

BACKGROUND: The von Willebrand factor (VWF) gene is a marker for spatial and temporal heterogeneity of the endothelium. A GATA motif at +220 has been implicated in basal VWF expression in vitro. Other studies have shown that GATA3 and VWF are transcriptionally downregulated in response to inflammatory mediators. OBJECTIVES: Our goal was to determine the importance of the +220 GATA motif in mediating expression of VWF promoter in vivo, and to elucidate whether the GATA element plays a role in spatial and/or temporal regulation of VWF expression. METHODS: ChIP and electrophoretic mobility shift assays were carried out in human umbilical vein endothelial cells (HUVEC). Reporter gene constructs containing 3.6 kb of the human VWF promoter with and without a mutation of the +220 GATA element were transfected into cultured endothelial cells or targeted to the Hprt locus of mice. The Hprt-targeted mice were subjected to endotoxemia. RESULTS: In protein-DNA binding assays, the +220 GATA motif bound GATA-2, -3 and -6. Mutation of the GATA site resulted in reduced basal promoter activity in HUVEC. When targeted to the Hprt locus of mice, the GATA mutation resulted in a significant, proportionate reduction of promoter activity in LacZ expressing vascular beds. Systemic administration of lipopolysaccharide (LPS) resulted in a widespread reduction in VWF mRNA expression and promoter activity. LPS-mediated repression of the VWF promoter was unaffected by the GATA mutation. CONCLUSIONS: A region of the VWF promoter between -2182 and the end of the first intron contains information for LPS-mediated gene repression. The +220 GATA motif is important for basal, but not LPS-repressible expression of the VWF gene.


Subject(s)
GATA Transcription Factors/physiology , Gene Expression Regulation/drug effects , Lipopolysaccharides/pharmacology , von Willebrand Factor/genetics , Animals , Cells, Cultured , Down-Regulation/genetics , Endothelium, Vascular/cytology , Humans , Hypoxanthine Phosphoribosyltransferase/genetics , Mice , Mice, Transgenic , Promoter Regions, Genetic , RNA, Messenger/analysis
8.
J Thromb Haemost ; 6(10): 1804-11, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18680538

ABSTRACT

BACKGROUND: Growth Arrest Specific gene product 6 (gas6) is a gamma-carboxylated protein that protects endothelial cells against apoptosis. Gas6 has previously been shown to induce phospatidyl-3-inositol-kinase (PI3K)/Akt signaling. Other studies have demonstrated a link between PI3K/Akt signaling and forkhead transcription factors in endothelial cells. OBJECTIVE: To test the hypothesis that gas6 promotes cell survival via a forkhead-dependent pathway. RESULTS AND CONCLUSIONS: Treatment of serum-starved human umbilical vein endothelial cells (HUVECs) with gas6 induced time-dependent phosphorylation and nuclear exclusion of FOXO1a. This effect was suppressed by the PI3K inhibitor wortmannin, demonstrating that FOXO1a phosphorylation is PI3-kinase dependent. Transduction of HUVECs with a phosphorylation-resistant form of FOXO1a [triple mutant (TM)-FOXO1a] abrogated the pro-survival effect of gas6 on serum-starved endothelial cells. Finally, treatment of serum-starved HUVECs with gas6 resulted in a reduction of FOXO1a transcriptional activity and downregulation of the pro-apoptotic gene, p27(kip1). Taken together, these findings suggest that gas6 protects endothelial cells from apoptosis by a mechanism that involves PI3K-Akt-dependent inactivation of FOXO1a.


Subject(s)
Endothelial Cells/metabolism , Forkhead Transcription Factors/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Active Transport, Cell Nucleus , Cell Survival , Cyclin-Dependent Kinase Inhibitor p27/genetics , Down-Regulation/genetics , Forkhead Box Protein O1 , Humans , Kinetics , Phosphatidylinositol 3-Kinases , Phosphorylation , Signal Transduction , Transcription, Genetic , Umbilical Veins/cytology
9.
J Thromb Haemost ; 5 Suppl 1: 283-91, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17635738

ABSTRACT

Hemostasis represents a finely tuned balance between procoagulant and anticoagulant forces. An imbalance of these forces may lead to clinically significant disease, including arterial, venous and/or microvascular thrombosis. The vast majority of hypercoagulable states are associated with local thrombus formation. The goal of this review is to discuss the mechanisms underlying site-specific thrombosis.


Subject(s)
Blood Vessels/pathology , Thrombosis/pathology , Humans
10.
J Thromb Haemost ; 3(7): 1392-406, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15892866

ABSTRACT

The endothelium is a highly metabolically active organ that is involved in many physiological processes, including the control of vasomotor tone, barrier function, leukocyte adhesion and trafficking, inflammation, and hemostasis. Endothelial cell phenotypes are differentially regulated in space and time. Endothelial cell heterogeneity has important implications for developing strategies in basic research, diagnostics and therapeutics. The goals of this review are to: (i) consider mechanisms of endothelial cell heterogeneity; (ii) discuss the bench-to-bedside gap in endothelial biomedicine; (iii) revisit definitions for endothelial cell activation and dysfunction; and (iv) propose new goals in diagnosis and therapy. Finally, these themes will be applied to an understanding of vascular bed-specific hemostasis.


Subject(s)
Endothelium, Vascular/anatomy & histology , Animals , Arteries/anatomy & histology , Capillaries/anatomy & histology , Cell Adhesion , Endothelial Cells/cytology , Humans , Leukocytes/cytology , Models, Anatomic , Phenotype , Time Factors , Veins/anatomy & histology
11.
J Thromb Haemost ; 1(2): 227-30, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12871493

ABSTRACT

Coagulation evolved as a means to stem the loss of blood and to defend against pathogens. The complexity of the clotting cascade has been cited as evidence for the existence of divine intervention. The objective of this review is to draw on the debate between creationists and evolutionary biologists to highlight important evolutionary principles that underlie the hemostatic mechanism. I propose the following: (a) as with all biological systems, the hemostatic mechanism displays non-linear complexity; (b) the cellular response represents primary hemostasis owing to its place in the evolutionary time scale and functional importance; and (c) the rapid evolution of the hemostatic mechanism in vertebrates is testimony to the power and versatility of gene duplications and exon shuffling.


Subject(s)
Biological Evolution , Blood Coagulation/genetics , Hemostasis , Animals , Blood Coagulation/physiology , Humans , Models, Biological , Time Factors
13.
J Biol Chem ; 276(50): 47632-41, 2001 Dec 14.
Article in English | MEDLINE | ID: mdl-11590177

ABSTRACT

The goal of this study was to delineate the transcriptional mechanisms underlying thrombin-mediated induction of vascular adhesion molecule-1 (VCAM-1). Treatment of human umbilical vein endothelial cells with thrombin resulted in a 3.3-fold increase in VCAM-1 promoter activity. The upstream promoter region of VCAM-1 contains a thrombin response element, two nuclear factor kappaB (NF-kappaB) motifs, and a tandem GATA motif. In transient transfection assays, mutation of the thrombin response element had no effect on thrombin induction. In contrast, mutation of either NF-kappaB site resulted in a complete loss of induction, whereas a mutation of the two GATA motifs resulted in a significant reduction in thrombin stimulation. In electrophoretic mobility shift assays, nuclear extracts from thrombin-treated endothelial cells displayed markedly increased binding to the tandem NF-kappaB and GATA motifs. The NF-kappaB complex was supershifted with anti-p65 antibodies, but not with antibodies to RelB, c-Rel, p50, or p52. The GATA complex was supershifted with antibodies to GATA-2, but not GATA-3 or GATA-6. A construct containing tandem copies of the VCAM-1 GATA motifs linked to a minimal thymidine kinase promoter was induced 2.4-fold by thrombin. Taken together, these results suggest that thrombin stimulation of VCAM-1 in endothelial cells is mediated by the coordinate action of NF-kappaB and GATA transcription factors.


Subject(s)
Endothelium/metabolism , NF-kappa B/metabolism , Thrombin/metabolism , Vascular Cell Adhesion Molecule-1/genetics , Amino Acid Motifs , Base Sequence , Cell Line , Cell Nucleus/metabolism , Cells, Cultured , DNA-Binding Proteins/metabolism , Dimerization , Dose-Response Relationship, Drug , GATA2 Transcription Factor , Humans , Luciferases/metabolism , Models, Genetic , Molecular Sequence Data , Mutation , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , RNA/metabolism , RNA, Messenger/metabolism , Ribonucleases/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Transcriptional Activation , Transfection , Tumor Necrosis Factor-alpha/metabolism , Umbilical Veins/cytology , Up-Regulation , Vascular Cell Adhesion Molecule-1/metabolism
14.
FASEB J ; 15(13): 2548-50, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11641265

ABSTRACT

Vascular endothelial growth factor (VEGF) is a potent vascular endothelial cell-specific mitogen that modulates endothelial cell function. In the present study, we show that VEGF induces manganese-superoxide dismutase (MnSOD) mRNA and protein in human coronary artery endothelial cells (HCAEC) and pulmonary artery endothelial cells. VEGF-mediated induction of MnSOD mRNA was inhibited by pretreatment with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI), and 4-(2-aminoethyl)-benzenesulfonyl fluoride, but not with the nitric oxide synthase inhibitor L-NAME (N-monomethyl-L-arginine) or the xanthine oxidase inhibitor allopurinol. VEGF stimulation of MnSOD was also inhibited by adenoviral-mediated overexpression of catalase Cu, Zn-SOD and a dominant-negative form of the small GTPase component of NADPH oxidase Rac1 (Rac1N17). Treatment of HCAEC with VEGF resulted in a transient increase in ROS production at 20 min, as measured by 2,7-dichlorodihydrofluorescein oxidation. This effect was abrogated by expression of Rac1N17. Taken together, these findings suggest that VEGF induces MnSOD by an NADPH oxidase-dependent mechanism and that VEGF signaling in the endothelium is coupled to the redox state of the cell.


Subject(s)
Endothelial Growth Factors/pharmacology , Endothelium, Vascular/drug effects , Lymphokines/pharmacology , NADPH Oxidases/metabolism , Superoxide Dismutase/drug effects , rac1 GTP-Binding Protein/physiology , Adenoviridae/genetics , Blotting, Northern , Blotting, Western , Catalase/genetics , Catalase/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Free Radical Scavengers/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Genetic Vectors/genetics , Humans , NADPH Oxidases/antagonists & inhibitors , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , rac1 GTP-Binding Protein/genetics
15.
Am J Physiol Gastrointest Liver Physiol ; 281(5): G1271-8, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11668036

ABSTRACT

Early growth response-1 (Egr-1) is a transcription factor that couples short-term changes in the extracellular milieu to long-term changes in gene expression. Under in vitro conditions, the Egr-1 gene has been shown to respond to many extracellular signals. In most cases, these findings have not been extended to the in vivo setting. The goal of the present study was to explore the role of epidermal growth factor (EGF) in mediating Egr-1 expression in hepatocytes under both in vitro and in vivo conditions. In HepG2 cells, Egr-1 protein and mRNA were upregulated in the presence of EGF. In stable transfections of HepG2 cells, a 1,200-bp Egr-1 promoter contained information for EGF response via a protein kinase C-independent, mitogen-activated protein kinase-dependent signaling pathway. A promoter region containing the two most proximal serum response elements was sufficient to transduce the EGF signal. In transgenic mice that carry the Egr-1 promoter coupled to the LacZ reporter gene, systemic delivery of EGF by intraperitoneal injection resulted in an induction of the endogenous Egr-1 gene and the Egr-1-lacZ transgene in hepatocytes. Together, these results suggest that the 1,200-bp promoter contains information for EGF response in hepatocytes both in vitro and in intact animals.


Subject(s)
DNA-Binding Proteins/genetics , Epidermal Growth Factor/pharmacology , Hepatocytes/drug effects , Hepatocytes/physiology , Immediate-Early Proteins , Promoter Regions, Genetic/physiology , Transcription Factors/genetics , Animals , Cells, Cultured , Early Growth Response Protein 1 , Gene Expression Regulation , Humans , Mice , Mice, Transgenic/genetics , Transgenes/drug effects , Transgenes/physiology
16.
Crit Care Med ; 29(7 Suppl): S28-34; discussion S34-5, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11445731

ABSTRACT

OBJECTIVES: To examine the role of vascular bed-specific pathways in determining the hemostatic phenotype in sepsis. DATA SOURCES/STUDY SELECTION: Published research and review articles related to hemostasis and endothelial cell biology. DATA EXTRACTION AND SYNTHESIS: The results of published studies have been used to generate a hypothesis of vascular bed-specific hemostasis in sepsis. CONCLUSIONS: In sepsis, coagulation is initiated by the extrinsic pathway and is amplified through the intrinsic pathway. In addition, the body's natural anticoagulant mechanisms are significantly dampened. Together, these changes result in a net imbalance of hemostasis. The nature of this imbalance varies from one vascular bed to the next according to the local set point of the endothelium. These concepts lay an important foundation for understanding the pathophysiology of sepsis.


Subject(s)
Blood Coagulation Disorders/microbiology , Endothelium, Vascular/physiology , Hemostasis/physiology , Sepsis/blood , Sepsis/complications , Animals , Apoptosis , Blood Coagulation Factors/physiology , Disease Models, Animal , Drug Design , Gene Expression Regulation/physiology , Humans , Phenotype , Receptor Cross-Talk/physiology , Sepsis/drug therapy , Sepsis/immunology , Sepsis/physiopathology , Signal Transduction/physiology , Thrombosis/etiology
17.
J Biol Chem ; 276(7): 5395-402, 2001 Feb 16.
Article in English | MEDLINE | ID: mdl-11098056

ABSTRACT

The angiogenic effects of vascular endothelial growth factor are mediated predominantly by the FLK-1/KDR receptor. An understanding of the transcriptional control mechanisms underlying flk-1/KDR expression should provide insight into the molecular basis of angiogenesis. In this study, we show that transforming growth factor-beta(1) (TGF-beta(1)) down-regulates expression of the endogenous flk-1/KDR gene in endothelial cells. In transient transfection assays, this effect was mapped to a palindromic GATA site in the 5'-untranslated region. In electrophoretic mobility shift assays, the palindromic GATA site was shown to bind to two molecules of GATA protein. Moreover, DNA-GATA interactions were inhibited by TGF-beta(1). Finally, in cotransfection assays, transactivation of the flk-1/KDR promoter by GATA-1 or GATA-2 was attenuated in TGF-beta(1)-treated cells. Taken together, these results suggest that the TGF-beta-1-mediated inhibition of the flk-1/KDR gene is mediated by a 5'-untranslated region palindromic GATA site.


Subject(s)
DNA-Binding Proteins/metabolism , Endothelium, Vascular/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Growth Factor/genetics , Transcription Factors/metabolism , Transforming Growth Factor beta/pharmacology , 5' Untranslated Regions , Animals , Cattle , Cell Line , Cells, Cultured , Consensus Sequence , DNA/metabolism , DNA Footprinting , Down-Regulation , Endothelium, Vascular/drug effects , GATA2 Transcription Factor , Humans , Promoter Regions, Genetic , RNA, Messenger/metabolism , Receptors, Vascular Endothelial Growth Factor , Transcriptional Activation/drug effects , Transforming Growth Factor beta1
18.
Semin Thromb Hemost ; 26(5): 463-78, 2000.
Article in English | MEDLINE | ID: mdl-11129402

ABSTRACT

Thrombosis reflects an imbalance between procoagulant and anticoagulant mechanisms. In some cases, thrombotic lesions are precipitated by gross changes in blood flow, vascular wall integrity, or systemic levels of coagulation factors. In other cases, thrombosis is induced by functional changes within the endothelium. Endothelial cells express a wide variety of factors that contribute to hemostasis, including procoagulants, anticoagulants, cell adhesion molecules, vasomotor substances, and cell survival signals. Because the endothelium displays a remarkable diversity of structure and function, the relative contribution of any one of these factors to the hemostatic balance varies between different vascular beds. In this review, we emphasize the heterogeneous nature of endothelial cell function. We then examine the role of endothelial diversity in modulating the phenotypic expression of thrombotic disorders.


Subject(s)
Endothelium, Vascular/physiology , Thrombosis/etiology , Animals , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Hemostasis , Humans , Thrombosis/pathology
19.
FEBS Lett ; 486(3): 252-6, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11119713

ABSTRACT

NADPH oxidase has been shown to play an important role in cardiovascular biology. The goal of the present study was to determine whether NADPH oxidase activity is important for endothelial cell growth and migration. In proliferation assays, growth factor- or serum-induced DNA synthesis in three different types of human endothelial cells was abrogated by inhibitors of NADPH oxidase, but not by inhibitors of xanthine oxidase or nitric oxide synthase. Moreover, vascular endothelial growth factor-induced migration of human endothelial cells was suppressed in the presence of NADPH oxidase inhibitors. These results support a potential role for NADPH oxidase in mediating angiogenesis.


Subject(s)
Endothelium, Vascular/metabolism , NADPH Oxidases/metabolism , Acetophenones/pharmacology , Allopurinol/pharmacology , Cell Division/drug effects , Cell Division/physiology , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured , Dose-Response Relationship, Drug , Endothelial Growth Factors/pharmacology , Endothelium, Vascular/cytology , Enzyme Inhibitors/pharmacology , Fluorescent Dyes , Humans , Lymphokines/pharmacology , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/pharmacology , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Sulfones/pharmacology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Xanthine Oxidase/antagonists & inhibitors
20.
FASEB J ; 14(13): 1870-2, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11023970

ABSTRACT

Egr-1 is an immediate early gene that couples short-term changes in the extracellular milieu to long-term changes in gene expression. Under in vitro conditions, the Egr-1 gene is expressed in many cell types and is induced by a wide variety of extracellular signals. The mechanisms by which the Egr-1 gene is regulated in vivo remain poorly understood. In this study, we have generated transgenic mice with a construct containing 1200 bp of the mouse Egr-1 promoter coupled to nuclear localized LacZ. In multiple independent lines of mice, reporter gene expression was detected in subsets of endothelial cells, vascular smooth-muscle cells, cardiomyocytes, neurons, and hepatocytes. This pattern closely resembled that of the endogenous gene. After partial hepatectomy, reporter gene activity was upregulated between two- and fivefold in regenerating livers. Taken together, these findings suggest that the Egr-1 promoter contains information for appropriate spatial and temporal expression in vivo.


Subject(s)
DNA-Binding Proteins/genetics , Immediate-Early Proteins/genetics , Promoter Regions, Genetic , Transcription Factors/genetics , Animals , Early Growth Response Protein 1 , Gene Expression Regulation , Genes, Reporter , Hepatectomy , Lac Operon , Mice , Mice, Transgenic , Tissue Distribution
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