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W C Aird

Publications and source records attributed to W C Aird.

10 recordsLinked to original sources
PubMed1999

Characterization of the mouse von Willebrand factor promoter.

Expression of the von Willebrand factor (vWF) gene is restricted to the endothelial and megakaryocyte lineages. Within the endothelium, expression of vWF varies between different vascular beds. We have previously shown that the human vWF promoter spanning a region between -2182 (relative to the start site of transcription) and the end of the first intron contains information for environmentally responsive, vascular bed-specific expression in the heart, skeletal muscle, and brain. In the present study, we cloned the mouse vWF (mvWF) promoter and studied its function in cultured endothelial cells and transgenic mice. In transient transfection assays, the mvWF gene was found to be regulated by distinct mechanisms in different endothelial cell subtypes. In independent lines of transgenic mice, an mvWF promoter fragment containing DNA sequences between -2645 and the end of the first intron directed endothelial cell-specific expression in the microvascular beds of the heart, brain, and skeletal muscle as well as the endothelial lining of the aorta. In 1 line of mice, reporter gene activity was also detected in bone marrow megakaryocytes. Taken together, these findings suggest that both the mouse and human vWF promoters are regulated by vascular bed-specific mechanisms.

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PubMed1999

A vascular bed-specific pathway.

The endothelial nitric oxide synthase (eNOS) gene is induced by a variety of extracellular signals under both in vitro and in vivo conditions. To gain insight into the mechanisms underlying environmental regulation of eNos expression, transgenic mice were generated with the 1,600-bp 5' flanking region of the human eNos promoter coupled to the coding region of the LacZ gene. In multiple independent lines of mice, transgene expression was detected within the endothelium of the brain, heart, skeletal muscle, and aorta. beta-galactosidase activity was consistently absent in the vascular beds of the liver, kidney, and spleen. In stable transfection assays of murine endothelial progenitor cells, the 1,600-bp promoter region was selectively induced by conditioned media from cardiac myocytes, skeletal myocytes, and brain astrocytes. Cardiac myocyte-mediated induction was partly abrogated by neutralizing anti-platelet-derived growth factor (PDGF) antibodies. In addition, promoter activity was upregulated by PDGF-AB. Analysis of promoter deletions revealed that a PDGF response element lies between -744 and -1,600 relative to the start site of transcription, whereas a PDGF-independent cardiac myocyte response element is present within the first 166 bp of the 5' flanking region. Taken together, these results suggest that the eNos gene is regulated in the cardiac endothelium by both a PDGF-dependent and PDGF-independent microvascular bed-specific signaling pathway.

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PubMed1998

PDGF mediates cardiac microvascular communication.

The diversity of cellular and tissue functions within organs requires that local communication circuits control distinct populations of cells. Recently, we reported that cardiac myocytes regulate the expression of both von Willebrand factor (vWF) and a transgene with elements of the vWF promoter in a subpopulation of cardiac microvascular endothelial cells (J. Cell Biol. 138:1117). The present study explores this communication. Histological examination of the cardiac microvasculature revealed colocalization of the vWF transgene with the PDGF alpha-receptor. Transcript analysis demonstrated that in vitro cardiac microvascular endothelial cells constitutively express PDGF-A, but not B. Cardiac myocytes induced endothelial expression of PDGF-B, resulting in PDGF-AB. Protein measurement and transcript analysis revealed that PDGF-AB, but not PDGF-AA, induced endothelial expression of vWF and its transgene. Antibody neutralization of PDGF-AB blocked the myocyte-mediated induction. Immunostaining demonstrated that vWF induction is confined to PDGF alpha-receptor-positive endothelial cells. Similar experiments revealed that the PDGF-AB/alpha-receptor communication also induces expression of vascular endothelial growth factor and Flk-1, critical components of angiogenesis. The existence of this communication pathway was confirmed in vivo. Injection of PDGF-AB neutralizing antibody into the amniotic fluid surrounding murine embryos extinguished expression of the transgene. In summary, these studies suggest that environmental induction of PDGF-AB/alpha-receptor interaction is central to the regulation of cardiac microvascular endothelial cell hemostatic and angiogenic activity.

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PubMed1998

Enhancement of murine cardiac chronotropy by the molecular transfer of the human beta2 adrenergic receptor cDNA.

Cardiac pacemaking offers a unique opportunity for direct gene transfer into the heart. An experimental system was developed to assay the effects of transferring the human beta2 adrenergic receptor (beta2AR) under in vitro, ex vivo, and finally in vivo conditions. Constructs encoding either beta2AR or LacZ were used in chronotropy studies with isolated myocytes, and transplanted as well as endogenous murine hearts. Murine embryonic cardiac myocytes were transiently transfected with plasmid constructs. The total percentage of myocytes spontaneously contracting was greater in beta2AR transfected cells, as compared with control cells (67 vs. 42+/-5%). In addition, the percentage of myocytes with chronotropic rates > 60 beats per minute (bpm) was higher in the beta2AR population, as compared with control cells (37 vs. 15+/-5%). The average contractile rate was greater in the beta2AR transfected myocytes at baseline (71+/-14 vs. 50+/-10 bpm; P < 0.001) as well as with the addition of 10(-)3 M isoproterenol (98+/-26 vs. 75+/-18 bpm; P < 0.05). Based on these results, a murine neonatal cardiac transplantation model was used to study the ex vivo effects of targeted expression of beta2AR. The constructs were transfected into the right atrium of transplanted hearts. Injection of the beta2AR construct increased the heart rate by approximately 40% (224+/-37 vs. 161+/-42 bpm; P < 0.005). Finally, the constructs were tested in vivo with injection into the right atrium of the endogenous heart. These results were similar to the ex vivo data with injection of the beta2AR constructs increasing the endogenous heart rates by approximately 40%, as compared with control injected hearts (550+/-42 vs. 390+/-37 bpm; P < 0.05). These studies demonstrate that local targeting of gene expression may be a feasible modality to regulate the cardiac pacemaking activity.

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PubMed1997

Vascular bed-specific expression of an endothelial cell gene is programmed by the tissue microenvironment.

The endothelium is morphologically and functionally adapted to meet the unique demands of the underlying tissue. At the present time, little is known about the molecular basis of endothelial cell diversity. As one approach to this problem, we have chosen to study the mechanisms that govern differential expression of the endothelial cell-restricted von Willebrand factor (vWF) gene. Transgenic mice were generated with a fragment of the vWF gene containing 2,182 bp of 5' flanking sequence, the first exon and first intron coupled to the LacZ reporter gene. In multiple independent lines of mice, beta-galactosidase expression was detected within endothelial cells in the brain, heart, and skeletal muscle. In isogeneic transplantation models, LacZ expression in host-derived auricular blood vessels was specifically induced by the microenvironment of the heart. In in vitro coculture assays, expression of both the transgene and the endogenous vWF gene in cardiac microvascular endothelial cells (CMEC) was upregulated in the presence of cardiac myocytes. In contrast, endothelial cell levels of thrombomodulin protein and mRNA were unchanged by the addition of ventricular myocytes. Moreover, CMEC expression of vWF was not influenced by the addition of 3T3 fibroblasts or mouse hepatocytes. Taken together, the results suggest that the vWF gene is regulated by vascular bed-specific pathways in response to signals derived from the local microenvironment.

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PubMed1996

Targeting of transgene expression to the vascular endothelium of mice by homologous recombination at the thrombomodulin locus.

We describe a straightforward gene-targeting technique to achieve uniform, stable, and genetically invariant expression of a transgene in the vascular endothelium of mice. To demonstrate the feasibility of this approach, the reporter gene bacterial beta-galactosidase was inserted via homologous recombination into the intronless thrombomodulin locus of murine embryonic stem cells. In this fashion, the lacZ gene is placed under the regulatory control of the endogenous thrombomodulin promoter. The expression of the transgene in adult mice recapitulated the widespread, stable, and high-level expression of the thrombomodulin gene in vascular endothelium. These data indicate that targeting of cDNAs into the thrombomodulin locus serves as a viable strategy to express transgenes in endothelial cells. Analysis of reporter gene expression revealed a heterogeneous pattern of thrombomodulin gene activity in the endothelium of the aorta and its tributaries. We also show that embryonic stem cells with a targeted thrombomodulin locus contribute in a mosaic fashion to the vascular endothelium of chimeric mice. This method for generating animals with a functionally heterogeneous cardiovascular system should provide an experimental technique for studying how localized genetic abnormalities in endothelial cell function lead to the development of vascular diseases.

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PubMed1996

Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos.

Embryonic lethality of thrombomodulin-deficient mice has indicated an essential role for this regulator of blood coagulation in murine development. Here, the embryonic expression pattern of thrombomodulin was defined by surveying beta-galactosidase activity in a mouse strain in which the reporter gene was placed under the regulatory control of the endogenous thrombomodulin promoter via homologous recombination in embryonic stem cells. The murine trophoblast was identified as a previously unrecognized anatomical site where TM expression is conserved between humans and mice and may exert a critical function during postimplantation development. Targeted reporter gene expression in mesodermal precursors of the endothelial cell lineage defined thrombomodulin as an early marker of vascular differentiation. Analysis of the thrombomodulin promoter in differentiating ES cells and in transgenic mice provided evidence for a disparate and cell type-specific gene regulatory control mechanism in the parietal yolk sac. The thrombomodulin promoter as defined in this study will allow the targeting of gene expression to the parietal yolk sac of transgenic mice and the initiation of investigations into the role of parietal endoderm in placental function.

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PubMed1995

Human von Willebrand factor gene sequences target expression to a subpopulation of endothelial cells in transgenic mice.

The present study was undertaken to define the 5' and 3' regulatory sequences of human von Willebrand factor gene that confer tissue-specific expression in vivo. Transgenic mice were generated bearing a chimeric construct that included 487 bp of 5' flanking sequence and the first exon fused in-frame to the Escherichia coli lacZ gene. In situ histochemical analyses in independent lines demonstrated that the von Willebrand factor promoter targeted expression of LacZ to a subpopulation of endothelial cells in the yolk sac and adult brain. LacZ activity was absent in the vascular beds of the spleen, lung, liver, kidney, testes, heart, and aorta, as well as in megakaryocytes. In contrast, in mice containing the lacZ gene targeted to the thrombomodulin locus, the 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside reaction product was detected throughout the vascular tree. These data highlight the existence of regional differences in endothelial cell gene regulation and suggest that the 733-bp von Willebrand factor promoter may be useful as a molecular marker to investigate endothelial cell diversity.

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PubMed1994

The interaction of GATA-binding proteins and basal transcription factors with GATA box-containing core promoters. A model of tissue-specific gene expression.

The core promoters of the rat platelet factor 4 (PF4), mouse erythropoietin and chicken beta globin genes contain a GATA motif in place of the consensus TATAAA site. In the case of the PF4 gene, this site has been shown to play a critical role in restricting transcription to the megakaryocyte lineage. In order to understand the mechanism of tissue specificity, we investigated the function of the GATA box-containing promoters in vitro. Our studies show that the TATA-binding protein of TFIID is required for initiation of transcription from the GATA box-containing promoters. GATA-1 interacts with the core promoter GATA motif and inhibits generation of preinitiation complexes. The functional significance of the inhibition of preinitiation complexes is supported by in vitro transcription assays in which transcription from the PF4 and erythropoietin core promoters is suppressed by GATA-1. We also demonstrate that GATA-2 inhibits initiation of transcription from the PF4 core promoter. Based on these results, we propose a model in which repression of PF4 expression in nonmegakaryocytes is mediated, in part, by competition between GATA-binding proteins and basal factors for the core promoter.

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