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Hypoxia Up-regulates Mouse Vascular Endothelial Growth Factor D Promoter Activity in Rat Pulmonary Microvascular Smooth-Muscle Cells* FREE TO VIEW

Xingwu Teng, PhD; Dechuan Li, MD, PhD; Roger A. Johns, MD
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*From the Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD.

Correspondence to: X. Teng, Anesthesiology and Critical Care Medicine, 600 North Wolfe St, Blalock 1415, Baltimore, MD 21287-4965

Chest. 2002;121(3_suppl):82S-83S. doi:10.1378/chest.121.3_suppl.82S
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Chronic hypoxia is implicated in the development of pulmonary hypertension; however, the mechanisms of the hypoxia-induced pulmonary hypertension are not clear. Vascular endothelial growth factor D (VEGF-D) is highly expressed in lung and plays a role in angiogenesis and vascular remodeling. Preliminary experiments using gene-array method showed that VEGF-D messenger RNA levels in the lung were significantly increased in mice exposed to 10% oxygen for 4 days. We hypothesize that hypoxia up-regulates VEGF-D and causes pulmonary hypertension through VEGF-D–mediated pulmonary vascular remodeling. To study the mechanism of up-regulation of VEGF-D by hypoxia, two VEGF-D promoter fragments, a 3,448-base pair (bp) fragment and a 523-bp fragment, were isolated from a mouse genomic library by genome walking. The fragments were sequenced (GenBank AF345635) and cloned into a pGL3 luciferase reporter vector. The reporter constructs were transfected into rat pulmonary microvascular smooth-muscle cells. Transfection efficiency was normalized by cotransfected pRL-TK renilla luciferase activity. Dual luciferase assays were performed after transfected rat pulmonary microvascular smooth-muscle cells were exposed to either 1% oxygen or 21% oxygen for 24 h. The luciferase activity was expressed as fold of the pGL3 basic activity. Hypoxia significantly increased the VEGF-D promoter activity. The activity of the 3,448-bp fragment was increased from 3.3 ± 0.08-fold to 7.5 ± 0.59-fold. The activity of the two colonies of the 523-bp fragment was increased from 2.9 ± 0.12-fold to 6.3 ± 0.36-fold and from 3.3 ± 0.08-fold to 7.5 ± 1.0-fold, respectively. Sequencing confirmed that the 523-bp fragment was located at 3′ end of the 3,448-bp fragment. The results suggest that hypoxia-induced VEGF-D expression is regulated in a region within the 523-bp fragment.

Abbreviations: bp = base pair; VEGF-D = vascular endothelial growth factor D




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