PMC

LacZ staining is reduced in Hprt-targeted mice carrying amutation of the +220 GATA motif. (A) Schematic representation of Hprt-VWF2-lacZ and Hprt-VWF2-GATAm-lacZ constructs. pA, polyadenylation. (B) Comparative whole mount LacZ stains of brain (a–c), heart (d–f) and skeletal muscle (diaphragm) (g–i) from Hprt-VWF2-lacZ (VWF-2) and Hprt-VWF2-GATAm-lacZ (VWF-2-GATAm) mice. All organs were harvested and stained in parallel.

Endotoxemia is associated with reduced VWF-2 and VWF-2GATAm promoter activity in vivo. Comparative whole mount LacZ stains of heart (A,D), skeletal muscle (diaphragm) (B,E) and brain (C,F) from Hprt-VWF2-lacZ (VWF-2) and Hprt-VWF2-GATAm-lacZ (VWF-2-GATAm) mice treated in the absence (LPS−) or presence (LPS+) of lipopolysaccharide (LPS) i.p. Organs were harvested and stained in parallel.

β-Galactosidase activity is reduced in Hprt-targeted mice carrying a mutation of the +220 GATA motif. β-Galactosidase activity of protein extracts from various organs of Hprt-VWF2-lacZ (VWF2) and Hprt-VWF2-GATAm-lacZ (VWF2-GATAm) mice treated in the absence or presence of lipopolysaccharide (LPS). Data are relative to reporter gene activity in untreated VWF2 mice. *P < 0.05;**P < 0.01.

Endotoxemia is associated with reduced von Willebrand factor (VWF) mRNA and protein expression in vivo. Wild-type male mice were injected i.p. with 18 mg/kg lipopolysaccharide (LPS) or saline (control, CTL). (A) Shown are results of quantitative real-time PCR analyses (mRNA copy number per 106 copies 18S) of VWF in brain, heart, lung, liver, kidney and spleen at 24 h. Data are expressed as mean + SD of three independent experiments. *P < 0.05, **P < 0.01 compared with untreated controls. (B) Double immunofluorescence staining for VWF and CD31 in the lung and heart. Arrows show endocardium. Scale bars = 150 μM. Sections were stained in parallel and were photographed using identical microscope settings.

The +220 GATA motif is important for basal expression of the von Willebrand factor (VWF)-2 promoter in primary human endothelial cells and binds multiple GATA factors. (A) Transient transfections of human umbilical vein endothelial cells (HUVEC) with VWF2-luciferase with or without mutation of the GATA motif. The results show the means and standard deviations of luciferase light units (relative to untreated cells) obtained in triplicate from at least three independent experiments. *P < 0.05. (B) ChIP assay for GATA binding to VWF +220 GATA motif in HUVEC. Bottom shows binding ratio relative to total input chromatin using each antibody in the ChIP reaction (mean of two independent experiments). (C) An electrophoretic mobility shift assay was performed with ³²P-labeled +220 GATA probe in the absence (lane 1) or presence of nuclear extract from HUVEC (lanes 2–8). In competition assays, a 50-fold molar excess of unlabeled wild-type (lane 3) or mutant (lane 4) +220 GATA probe was added to the reaction mixture. Alternatively, nuclear extract was incubated in the presence of antibodies against GATA2 (lane 5), GATA3 (lane 6), GATA6 (lane 7) or control antibody (lane 8). The arrow indicates a specific DNA–protein complex. The results are representative of three independent experiments.