Specific bone morphogenic protein receptor II mutations found in primary pulmonary hypertension cause different biochemical phenotypes in vitro.

tion 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 0.08fold 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 hypoxiainduced VEGF-D expression is regulated in a region within the 523-bp fragment.