Hypoxia up-regulates angiopoietin-2, a Tie-2 ligand, in mouse mesangial cells

Abstract

Angiopoietins are secreted factors modulating endothelial survival and morphogenesis. Our previous studies demonstrated angiopoietin-2 (Ang-2) promoter activity in vivo in maturing kidney vascular smooth muscle and mesangial cells, with Tie-2 expressed by adjacent endothelia, including glomerular capillaries. In this study we investigated Ang-2 expression in immortalized mouse mesangial cell lines and studied the response to hypoxia. Using reverse transcription-polymerase chain reaction, Ang-2 and Ang-3 mRNA were detected but Ang-1 and Tie-2 transcripts were absent. As assessed by Northern and slot blotting, 8 to 24 hours hypoxia (3% O(2)) significantly increased Ang-2 mRNA levels versus normoxic (21% O(2)) cells and the rate of Ang-2 mRNA degradation was similar in both conditions, consistent with increased transcription. Hypoxia also increased immunoreactive Ang-2 in cell lysates. Hypoxic stimulation of Ang-2 mRNA was significantly reduced by inhibitors of tyrosine kinase (genistein) and protein kinase C (GF109203X), but not by a mitogen-activated protein kinase 1 inhibitor (PD98059). Furthermore, hypoxia coincidentally up-regulated levels of vascular endothelial growth factor (VEGF) mRNA in these cells. Finally, in vivo, immunoreactive Ang-2 was observed in the cores of immature glomeruli of neonatal mice, but immunostaining in this location was absent in four-week postnatal mice. This is the first demonstration that isolated mesangial cells express Ang-2 mRNA and protein and up-regulate Ang-2 in response to hypoxia. We speculate that hypoxia-induced, mesangial-derived Ang-2 and VEGF may have synergistic paracrine roles in the growth of glomerular endothelia during normal development and diseases.