Abstract 415: Sepiapterin Improves the Angiogenesis in Pulmonary Artery Endothelial Cells from Developing Lungs with In Utero Pulmonary Hypertension
Tetrahydrobiopterin (BH4) is an essential cofactor for eNOS. Sepiapterin (Sep) is converted into BH4 by the salvage pathway. BH4 is labile at physiological pH and easily oxidized to BH3 or BH2, making it useless as a cofactor for eNOS dependent •NO generation. Loss of BH4, whether through increased oxidation or impaired synthesis, has been linked to pulmonary hypertension. Previously we showed that NADPH oxidase derived superoxide (O2•−) impaired angiogenesis of Pulmonary Artery Endothelial Cells (PAEC) isolated from in utero pulmonary hypertension fetal lambs (HTFL). As increased NADPH oxidase activity has been linked to oxidation of BH4 and in turn, impaired eNOS activity, we hypothesized that restoring BH4 with Sep might improve PAEC function isolated from HTFL. To test this hypothesis we supplemented PAEC isolated from normotensive fetal lambs and HTFL with Sep (30 μM) and then examined its effects on angiogenesis. Angiogenesis was quantified with respect to tube length, cell invasion, decrease in the gap between cut edges of a PAEC monolayer and % of apoptotic cells. Sep increased tube length in PAEC from HTFL from 205.6±77.5 μm/HPF to 670.4±163.5 μm/HPF, 548.1±231.1 μm/HPF to 1753.7±454.0 μm/HPF, and 21.9±24.4 μm/HPF to 231.5±93.6 μm/HPF after 8, 14 and 24 hours incubation, respectively (p<0.05). Sep increased the number of cells invading transwells in PAEC from HTFL cultures from 60.7±14.2/HPF to 101.8±13.0/HPF (p=0.002) at 24 hours. Sep decreased the gap in PAEC from HTFL from 170.3±59.9 μm to 15.9±22.6 μm (p<0.001) at 24 hours. Finally, Sep decreased the % of apoptotic cells in PAEC cultures from HTFL from 18.6±6.1 to 6.6±2.2 (p=0.0005). No significant changes in angiogenic responses of PAEC isolated from normo-tensive fetal lungs were observed with Sep treatment. Taken together Sep improves angiogenic responses of HTFL PAEC in vitro without altering angiogenic responses of normal PAEC. As impaired angiogenesis is one of the hallmarks of pulmonary hypertension in fetal lambs these data suggest that Sep treatments may be important for increasing angiogenesis and improving clinical management of neonates with pulmonary hypertension.