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William C Aird

Publications and source records attributed to William C Aird.

At least 37 records · Page 2Linked to original sources
PubMed2004

Endothelial cells present antigens in vivo.

BACKGROUND: Immune recognition of vascular endothelial cells (EC) has been implicated in allograft rejection, protection against pathogens, and lymphocyte recruitment. However, EC pervade nearly all tissues and predominate in none, complicating any direct test of immune recognition. Here, we examined antigen presentation by EC in vivo by testing immune responses against E. coli beta-galactosidase (beta-gal) in two lines of transgenic mice that express beta-gal exclusively in their EC. TIE2-lacZ mice express beta-gal in all EC and VWF-lacZ mice express beta-gal in heart and brain microvascular EC. RESULTS: Transgenic and congenic wild type FVB mice immunized with beta-gal expression vector DNA or beta-gal protein generated high titer, high affinity antisera containing comparable levels of antigen-specific IgG1 and IgG2a isotypes, suggesting equivalent activation of T helper cell subsets. The immunized transgenic mice remained healthy, their EC continued to express beta-gal, and their blood vessels showed no histological abnormalities. In response to beta-gal in vitro, CD4+ and CD8+ T cells from immunized transgenic and FVB mice proliferated, expressed CD25, and secreted IFN-gamma. Infection with recombinant vaccinia virus encoding beta-gal raised equivalent responses in transgenic and FVB mice. Hearts transplanted from transgenic mice into FVB mice continued to beat and the graft EC continued to express beta-gal. These results suggested immunological ignorance of the transgene encoded EC protein. However, skin transplanted from TIE2-lacZ onto FVB mice lost beta-gal+ EC and the hosts developed beta-gal-specific antisera, demonstrating activation of host immune effector mechanisms. In contrast, skin grafted from TIE2-lacZ onto VWF-lacZ mice retained beta-gal+ EC and no antisera developed, suggesting a tolerant host immune system. CONCLUSION: Resting, beta-gal+ EC in transgenic mice tolerize specific lymphocytes that would otherwise respond against beta-gal expressed by EC within transplanted skin. We conclude that EC effectively present intracellular "self" proteins to the immune system. However, antigen presentation by EC does not delete or anergize a large population of specific lymphocytes that respond to the same protein following conventional immunization with protein or expression vector DNA. These results clearly demonstrate striking context sensitivity in the immune recognition of EC, a subtlety that must be better understood in order to treat immune diseases and complications involving the vasculature.

Animals↗
PubMed2004

Interaction between hex and GATA transcription factors in vascular endothelial cells inhibits flk-1/KDR-mediated vascular endothelial growth factor signaling.

Recent evidence supports a role for GATA transcription factors as important signal intermediates in differentiated endothelial cells. The goal of this study was to identify proteins that interact with endothelial-derived GATA transcription factors. Using yeast two-hybrid screening, we identified hematopoietically expressed homeobox (Hex) as a GATA-binding partner in endothelial cells. The physical association between Hex and GATA was confirmed with immunoprecipitation in cultured cells. Hex overexpression resulted in decreased flk-1/KDR expression, both at the level of the promoter and the endogenous gene, and attenuated vascular endothelial growth factor-mediated tube formation in primary endothelial cell cultures. In electrophoretic mobility shift assays, Hex inhibited the binding of GATA-2 to the flk-1/KDR 5'-untranslated region GATA motif. Finally, in RNase protection assays, transforming growth factor beta1, which has been previously shown to decrease flk-1 expression by interfering with GATA binding activity, was shown to increase Hex expression in endothelial cells. Taken together, the present study provides evidence for a novel association between Hex and GATA and suggests that transforming growth factor beta-mediated repression of flk-1/KDR and vascular endothelial growth factor signaling involves the inducible formation of inhibitory Hex-GATA complexes.

Cell Line↗
PubMed2004

Natural anticoagulant inhibitors: activated Protein C.

Protein C is a vitamin-K-dependent zymogen, whose congenital deficiency state leads to increased risk for venous thrombosis. Activated Protein C (aPC) exerts its anticoagulant function by inhibiting the cofactors in the clotting cascade, Factors Va and VIIIa. In addition, aPC displays anti-inflammatory, anti-apoptotic and profibrinolytic activities. A recombinant form of human aPC (rhAPC) is the first drug reported to improve survival in patients with severe sepsis. The major toxicity associated with treatment is bleeding. Appropriate use of rhAPC depends on an understanding of its mechanisms of action and risk:benefit profile. The goals of this review are: to describe the Protein C pathway; to discuss the definitions, epidemiology and pathophysiology of severe sepsis; to provide a conceptual framework for understanding the role of rhAPC in this syndrome; and to address frequently asked questions about the day-to-day use of this agent.

Anticoagulants↗
PubMed2004

Endothelium as an organ system.

The endothelium is a highly dynamic cell layer that is involved in a multitude of physiologic functions, including the control of vasomotor tone, the trafficking of cells and nutrients, the maintenance of blood fluidity, and the growth of new blood vessels. Over the past several decades, advances in basic research of the endothelium have far outstripped those in the clinic. One explanation for this growing bench-to-bedside chasm relates to the inflexible and largely outdated nature of the present-day medical infrastructure. The constraints of medical subspecialization have created a conceptual blind spot, namely, the inability to appreciate the endothelium for what it is: a cell layer that is teeming with life, every bit as active as any other organ in the body. The overall goal of this review is to bring the endothelium "to life" and to argue that future breakthroughs in biomedicine are contingent on acceptance of the endothelium as a bona fide organ system.

Animals↗
PubMed2003

Vascular endothelial growth factor activates PI3K/Akt/forkhead signaling in endothelial cells.

OBJECTIVE: Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor that promotes endothelial cell (EC) survival, migration, and permeability. The forkhead transcription factors FKHR, FKHRL1, and AFX are mammalian orthologues of DAF-16, a forkhead protein that controls longevity in Caenorhabditis elegans. In this study, we examined whether VEGF is coupled to phosphatidyl inositol 3-kinase (PI3K)/Akt/forkhead in ECs. METHODS AND RESULTS: We demonstrate that human ECs express members of the forkhead family (FKHR, FKHRL1, and AFX) and that VEGF modulates the phosphorylation, subcellular localization, and transcriptional activity of one or more of these isoforms by a PI3K/Akt signaling pathway. VEGF inhibited EC apoptosis, promoted DNA synthesis and the G(1)-to-S transition, and reduced expression of the cyclin-dependent kinase inhibitor p27(kip1). Each of these effects was blocked by the PI3K inhibitor LY294002 or by a phosphorylation-resistant mutant of FKHRL1, but not by wild-type FKHRL1. CONCLUSIONS: These results suggest that VEGF signaling in ECs is coupled to forkhead transcription factors through a PI3K/Akt-dependent pathway.

Active Transport, Cell Nucleus↗
PubMed2003

Thrombin and phenotypic modulation of the endothelium.

Thrombin signaling in the endothelium is linked to multiple phenotypic changes, including alterations in permeability, vasomotor tone, and leukocyte trafficking. The thrombin signal is transduced, at least in part, at the level of gene transcription. In this review, we focus on the role of thrombin signaling and transcriptional networks in mediating downstream gene expression and endothelial phenotype. In addition, we report the results of DNA microarrays in control and thrombin-treated endothelial cells. We conclude that (1) thrombin induces the upregulation and downregulation of multiple genes in the endothelium, (2) thrombin-mediated gene expression involves a multitude of transcription factors, and (3) future breakthroughs in the field will depend on a better understanding of the spatial and temporal dynamics of these transcriptional networks.

Blood Coagulation↗
PubMed2003

The Akt-regulated forkhead transcription factor FOXO3a controls endothelial cell viability through modulation of the caspase-8 inhibitor FLIP.

FLICE-inhibitory protein (FLIP) is a homolog of caspase-8 that lacks catalytic activity and has been shown to be important in protecting endothelial cells from apoptosis. The serine/threonine kinase Akt/PKB was recently reported to promote FLIP expression in endothelial and tumor cells. Here we examined the role of the forkhead transcription factor FOXO3a, a downstream target of Akt, in controlling FLIP regulation in endothelial cells. FOXO3a nuclear translocation was regulated by Akt in human umbilical vein endothelial cells. Transduction of a nonphosphorylatable, constitutively active mutant of FOXO3a (TM-FOXO3a) led to the down-regulation of FLIP levels. Transduction with TM-FOXO3a also increased caspase-8 activity and promoted apoptosis in endothelial cells. Conversely, transduction of a dominant-negative mutant of FOXO3a up-regulated FLIP levels and protected endothelial cells from apoptosis under serum deprivation conditions. Restoration of intracellular FLIP blocked caspase-8 activation and inhibited apoptosis in TM-FOXO3a-transduced cells. These data suggest that FOXO3a is a downstream target of Akt in endothelial cells that can promote apoptosis via FLIP down-regulation and activation of the extrinsic apoptotic pathway.

Active Transport, Cell Nucleus↗
PubMed2003

Ets motifs are necessary for endothelial cell-specific expression of a 723-bp Tie-2 promoter/enhancer in Hprt targeted transgenic mice.

OBJECTIVE: Tie-2 is an endothelial cell-specific receptor tyrosine kinase that is involved in the remodeling of blood vessels and angiogenesis. Our goal was to characterize Tie-2 promoter function as a means of providing insight into the mechanisms of endothelial cell-specific gene regulation. METHODS AND RESULTS: When targeted to the Hprt locus of mice, a small Tie-2 promoter fragment (containing a 300-bp intronic enhancer coupled upstream to a 423-bp core promoter) (T-short) directed widespread endothelial cell expression in vivo. The T-short promoter contains 2 clusters of Ets sites, one in the first exon, the other in the intronic enhancer. In cultured endothelial cells, a combined mutation of the Ets motifs resulted in a significant reduction in promoter activity. Consistent with these results, the same Ets mutations resulted in a loss of detectable expression of the T-short promoter in all vascular beds with the notable exception of the brain. CONCLUSIONS: These results suggest that the T-short promoter contains information for widespread expression in the vascular tree, Ets sites are necessary for in vivo promoter activity, and the shorter Tie-2 fragment may be useful as a tool to direct heterologous gene expression within the intact endothelium.

Amino Acid Motifs↗
PubMed2003

The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome.

Severe sepsis, defined as sepsis with acute organ dysfunction, is associated with high morbidity and mortality rates. The development of novel therapies for sepsis is critically dependent on an understanding of the basic mechanisms of the disease. The pathophysiology of severe sepsis involves a highly complex, integrated response that includes the activation of a number of cell types, inflammatory mediators, and the hemostatic system. Central to this process is an alteration of endothelial cell function. The goals of this article are to (1) provide an overview of sepsis and its complications, (2) discuss the role of the endothelium in orchestrating the host response in sepsis, and (3) emphasize the potential value of the endothelium as a target for sepsis therapy.

Endothelium, Vascular↗
PubMed2003

Regulation of tissue factor and inflammatory mediators by Egr-1 in a mouse endotoxemia model.

In septic shock, tissue factor (TF) activates blood coagulation, and cytokines and chemokines orchestrate an inflammatory response. In this study, the role of Egr-1 in lipopolysaccharide (LPS) induction of TF and inflammatory mediators in vivo was evaluated using Egr-1(+/+) and Egr-1(-/-) mice. Administration of LPS transiently increased the steady-state levels of Egr-1 mRNA in the kidneys and lungs of Egr-1(+/+) mice with maximal induction at one hour. Egr-1 was expressed in epithelial cells in the kidneys and lungs in untreated and LPS-treated mice. LPS induction of monocyte chemoattractant protein mRNA in the kidneys and lungs of Egr-1(-/-) mice was not affected at 3 hours, but its expression was significantly reduced at 8 hours compared with the expression observed in Egr-1(+/+) mice. Similarly, LPS induction of TF mRNA expression in the kidneys and lungs at 8 hours was reduced in Egr-1(-/-) mice. However, Egr-1 deficiency did not affect plasma levels of tumor necrosis factor alpha in endotoxemic mice. Moreover, Egr-1(+/+) and Egr-1(-/-) mice exhibited similar survival times in a model of acute endotoxemia. These data indicate that Egr-1 does not contribute to the early inflammatory response in the kidneys and lungs or the early systemic inflammatory response in endotoxemic mice. However, Egr-1 does contribute to the sustained expression of inflammatory mediators and to the maximal expression of TF at 8 hours in the kidneys and lungs.

Animals↗
PubMed2003

Endothelial cell heterogeneity.

OBJECTIVE: To review recent advances in the field of endothelial cell heterogeneity, and to apply this knowledge to an understanding of site-specific vasculopathy, including acute lung injury. DATA SOURCES AND STUDY SELECTION: Published research and review articles in the English language related to endothelial cell biology and endothelial cell heterogeneity. DATA EXTRACTION AND SYNTHESIS: The results of published studies have been used to provide a perspective of endothelial cell phenotypes in health and disease. CONCLUSIONS: The structure and function of endothelial cells are differentially regulated in space and time. Far from being a giant monopoly of homogeneous cells, the endothelium represents a consortium of smaller enterprises of cells located within blood vessels of different tissues. Although united in certain functions, each enterprise is uniquely adapted to meet the demands of the underlying tissue. The endothelium may also vary in its response to pathophysiologic stimuli and therefore contribute to the focal nature of vasculopathic disease states. In acute lung injury, the unique properties of the endothelium may conspire with systemic imbalances to localize pathology to the pulmonary vasculature.

Animals↗
PubMed2003

Platelet-endothelial interactions: sepsis, HIT, and antiphospholipid syndrome.

Acquired abnormalities in platelets, endothelium, and their interaction occur in sepsis, immune heparin-induced thrombocytopenia (HIT), and the antiphospholipid syndrome. Although of distinct pathogeneses, these three disorders have several clinical features in common, including thrombocytopenia and the potential for life- and limb-threatening thrombotic events, ranging from microvascular (sepsis > antiphospholipid > HIT) to macrovascular (HIT > antiphospholipid > sepsis) thrombosis, both venous and arterial. In Section I, Dr. William Aird reviews basic aspects of endothelial-platelet interactions as a springboard to considering the common problem of thrombocytopenia (and its mechanism) in sepsis. The relationship between thrombocytopenia and other aspects of the host response in sepsis, including activation of coagulation/inflammation pathways and the development of organ dysfunction, is discussed. Practical issues of platelet count triggers and targeted use of activated protein C concentrates are reviewed. In Section II, Dr. Theodore Warkentin describes HIT as a clinicopathologic syndrome, i.e., the diagnosis should be based on the concurrence of an appropriate clinical picture together with detection of platelet-activating and/or platelet factor 4-dependent antibodies (usually in high levels). HIT is a profound prothrombotic state (odds ratio for thrombosis, 20-40), and the risk for thrombosis persists for a time even when heparin is stopped. Thus, pharmacologic control of thrombin (or its generation), and postponing oral anticoagulation pending substantial resolution of thrombocytopenia, is appropriate. Indeed, coumarin-associated protein C depletion during uncontrolled thrombin generation of HIT can explain limb loss (coumarin-associated venous limb gangrene) or skin necrosis syndromes in some patients. In Section III, Dr. Jacob Rand presents the most recent concepts on the mechanisms of thrombosis in the antiphospholipid syndrome, and focuses on the role of beta(2)-glycoprotein I as a major antigenic target in this condition. Diagnosis of the syndrome is often complicated because the clinical laboratory tests to identify this condition have been empirically derived. Dr. Rand addresses the practical aspects of current testing for the syndrome and current recommendations for treating patients with thrombosis and with spontaneous pregnancy losses.

Antiphospholipid Syndrome↗
PubMed2003

The hematologic system as a marker of organ dysfunction in sepsis.

Sepsis with acute organ dysfunction (severe sepsis) results from a systemic proinflammatory and procoagulant response to infection. Organ dysfunction in the patient with sepsis is associated with increased mortality. Although most organs have discrete anatomical boundaries and carry out unified functions, the hematologic system is poorly circumscribed and serves several unrelated functions. This review addresses the hematologic changes associated with sepsis and provides a framework for prompt diagnosis and rational drug therapy. Data sources used include published research and review articles in the English language related to hematologic alterations in animal models of sepsis and in critically ill patients. Hematologic changes are present in virtually every patient with severe sepsis. Leukocytosis, anemia, thrombocytopenia, and activation of the coagulation cascade are the most common abnormalities. Despite theoretical advantages of using granulocyte colony-stimulating factor to enhance leukocyte function and/or circulating numbers, large clinical trials with these growth factors are lacking. Recent studies support a reduction in the red blood cell transfusion threshold and the use of erythropoietin treatment to reduce transfusion requirements. Treatment of thrombocytopenia depends on the cause and clinical context but may include platelet transfusions and discontinuation of heparin or other inciting drugs. The use of activated protein C may provide a survival benefit in subsets of patients with severe sepsis. The hematologic system should not be overlooked when assessing a patient with severe sepsis. A thorough clinical evaluation and panel of laboratory tests that relate to this organ system should be as much a part of the work-up as taking the patient's blood pressure, monitoring renal function, or measuring liver enzymes.

Blood Transfusion↗
PubMed2002

Thrombin stimulation of vascular adhesion molecule-1 in endothelial cells is mediated by protein kinase C (PKC)-delta-NF-kappa B and PKC-zeta-GATA signaling pathways.

We recently demonstrated that thrombin induces the expression of vascular adhesion molecule-1 (VCAM-1) in endothelial cells by an NF-kappaB- and GATA-dependent mechanism. In the present study, we describe the signaling pathways that mediate this response. Thrombin stimulation of the VCAM-1 gene and promoter in human umbilical vein endothelial cells was inhibited by preincubation with the phosphatidylinositol 3-kinase inhibitor, LY294002, the protein kinase C (PKC)-delta inhibitor, rottlerin, a PKC-zeta peptide inhibitor, or by overexpression of dominant negative (DN)-PKC-zeta. In electrophoretic mobility shift assays, thrombin-mediated induction of NF-kappaB p65 binding to two NF-kappaB motifs in the upstream promoter region of VCAM-1 was blocked by LY294002 and rottlerin, whereas the inducible binding of GATA-2 to a tandem GATA motif was inhibited by LY294002 and the PKC-zeta peptide inhibitor. In co-transfection assays, thrombin stimulation of a minimal promoter containing multimerized VCAM-1 NF-kappaB sites was inhibited by DN-PKC-delta but not DN-PKC-zeta. In contrast, thrombin-mediated transactivation of a minimal promoter containing tandem VCAM-1 GATA motifs was inhibited by DN-PKC-zeta but not DN-PKC-delta. Finally, thrombin failed to induce VCAM-1 expression in vascular smooth muscle cells. Taken together, these data suggest that the endothelial cell-specific effect of thrombin on VCAM-1 expression involves the coordinate activity of PKC-delta-NF-kappaB and PKC-zeta-GATA signaling pathways.

Acetophenones↗
PubMed2002

Differential regulation of the von Willebrand factor and Flt-1 promoters in the endothelium of hypoxanthine phosphoribosyltransferase-targeted mice.

An important limitation of standard transgenic assays is that multiple copies of the transgene are inserted randomly into the mouse genome, resulting in line-to-line variation in expression. One way to control for these variables is to target a single copy of the transgene to a defined locus of the mouse genome by homologous recombination. In the present study, we have used such an approach to target the promoters of 2 different genes, namely von Willebrand factor (VWF) and Flt-1, to the hypoxanthine phosphoribosyltransferase (Hprt) gene locus. Consistent with previous findings in standard transgenic animals, we report that the VWF promoter contains information for expression in a subset of endothelial cells in the heart, skeletal muscle, and brain. In contrast, the Flt-1 promoter directs expression in all vascular beds except for the liver. The Flt-1 transgene was active in the endothelium of tumor xenografts, whereas the VWF promoter was not. Under in vitro conditions, conditioned medium from tumor cells resulted in a significant up-regulation of Flt-1 mRNA and promoter activity, but no change in VWF levels. Taken together, these results suggest that (1) Hprt locus targeting is a valuable tool for studying vascular bed-specific gene regulation, (2) the VWF and Flt-1 promoters are regulated by distinct transcriptional mechanisms in the intact endothelium, and (3) tumor angiogenesis results in the differential activation of endothelial cell-specific promoters.

Animals↗
PubMed2002

The proximal serum response element in the Egr-1 promoter mediates response to thrombin in primary human endothelial cells.

Thrombin signaling in endothelial cells provides an important link between coagulation and inflammation. We report here that thrombin induces endogenous Egr-1 mRNA and Egr-1 promoter activity in primary human endothelial cells by approximately 6-fold and 3-fold, respectively. In transient transfection assays, deletion of the 3' cluster of serum response elements (SREs), but not the 5' cluster of SREs, resulted in a loss of thrombin response. When coupled to a heterologous core promoter, a region spanning the 3' SRE cluster contained information for thrombin response, whereas a region spanning the 5' SRE cluster had no such effect. A point mutation of the most proximal SRE (SRE-1), but not of the proximal Ets motif or upstream SREs, abrogated the response to thrombin. In electrophoretic mobility shift assays, nuclear extracts from thrombin-treated cells displayed increased binding of total and phosphorylated serum response factor (SRF) to SRE-1. Thrombin-mediated induction of Egr-1 was blocked by inhibitors of MEK1/2, but not by inhibitors of protein kinase C, phosphatidylinositol 3-kinase, or p38 mitogen-activated protein kinase (MAPK). Taken together, these data suggest that thrombin induces Egr-1 expression in endothelial cells by a MAPK-dependent mechanism that involves an interaction between SRF and SRE-1.

Amino Acid Motifs↗