Metformin upregulates the expression of Gli1 in vascular endothelial cells in hyperoxia-exposed neonatal mice.

Bronchopulmonary dysplasia (BPD) is characterized by arrested alveolar and vascular development in premature infants. Metformin protects against the cardiovascular impairment induced by diabetes. The aim of this study was to investigate whether metformin could also enhance pulmonary vascular development in hyperoxic neonatal mice and investigate possible mechanisms involved. C57BL/6J newborn mice were randomly assigned to either of two groups - the room air group or the hyperoxia group - within 12 h postnatally. The mice were subcutaneously injected with metformin (100 mg/kg) or saline for 14 days. Lung morphology and PECAM-1 (CD31) expression in the lung were evaluated at postnatal days 7 and 14. Ki-67 and Gli1 expression in vascular endothelial cells was evaluated at postnatal day 14 by immunofluorescence staining. Flow cytometry (FCM) was also used to analyze Gli1 expression. Human umbilical vein endothelial cell (HUVECs) were used to investigate the role of metformin in vascular proliferation and tubular formation under 90% oxygen in vitro by cell counting Kti-8 (CCK8) assays and tube formation assays. Exposure to hyperoxia resulted in impaired lung development in newborn mice. Metformin enhanced the terminal airspace and radial alveolar count in newborn mice thus exposed. Immunohistochemistry staining and western blot assays revealed that metformin enhanced the expression of CD31 in hyperoxia-exposed newborn mice. Immunofluorescence staining showed that metformin enhanced the expression of Ki-67 in vascular endothelial cells. Furthermore, both immunofluorescence staining and FCM demonstrated that metformin increased Gli1 expression in vascular endothelial cells. Additionally, cell counting Kit-8 (CCK8) and viability assays of HUVECs in vitro both indicated that metformin improved the vascular proliferation and tube formation of HUVECs under 90% oxygen. These results indicated that metformin enhanced lung vascular development and upregulated the expression of Gli1 in the pulmonary vascular endothelial cells in hyperoxic neonatal mice.