Vascular endothelial growth factor mediates reactive angiogenesis in the postnatal developing brain.

Although chronic sublethal hypoxia has been shown to promote angiogenesis in the developing brain, the pathogenesis of this response is unknown. We hypothesized that this response may be mediated in part by vascular endothelial growth factor (VEGF). We reared newborn rats (P3) in a chamber with FIO2 of 9.5 +/- 1% (exposed, E). At P33, the animals were removed from the chamber and the brains prepared for immunohistochemistry, mRNA extraction, or horseradish peroxidase (HRP) permeability studies. We also isolated beagle brain germinal matrix endothelial cells from PND 1 beagle pups and placed them in three-dimensional (3-D) coculture with PND 1 rat forebrain astrocytes. Cultures were grown for 6 days in 11% O2 and compared to control 3-D cocultures. When compared to age-matched controls, the experimental rats had significantly increased cortical vascular density (vessels/mm2: 518 +/- 18 vs. 400 +/- 15, P = 0.025). HRP studies demonstrated significantly increased permeability in all cortical vessels examined in experimental rats compared to controls. Compared to controls, VEGF mRNA from hypoxic pups was increased 2.4 times, and immunohistochemical studies of VEGF protein confirmed this finding. Similarly, when compared to controls, hypoxic cocultures of brain microvascular endothelial cells and astrocytes demonstrated significant increase in tubelike structures representing in vitro angiogenesis. Additionally, astrocyte VEGF protein levels increased 4.4-fold in hypoxic compared to control astrocyte cultures and VEGF protein levels increased 1.7-fold in hypoxic compared to control cocultures. Finally, addition of VEGF (10 ng/ml culture medium) to BBMEC alone in 3-D culture elicited not only significant proliferation (P = 0.001) but also increased tube formation. These data demonstrate that the developing brain responds to chronic sublethal hypoxia with increases in permeability and angiogenesis and suggest that VEGF mediates this response.