Roles of HIFs and VEGF in angiogenesis in the retina and brain
Amir Rattner, John Williams, Jeremy Nathans
Amir Rattner, John Williams, Jeremy Nathans
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Research Article Development Vascular biology

Roles of HIFs and VEGF in angiogenesis in the retina and brain

Abstract

Vascular development in the mammalian retina is a paradigm for CNS vascular development in general, and its study is revealing fundamental mechanisms that explain the efficacy of antiangiogenic therapies in retinal vascular disease. During development of the mammalian retina, hypoxic astrocytes are hypothesized to secrete VEGF, which attracts growing endothelial cells as they migrate radially from the optic disc. However, published tests of this model using astrocyte-specific deletion of Vegf in the developing mouse retina appear to contradict this theory. Here, we report that selectively eliminating Vegf in neonatal retinal astrocytes with a Gfap-Cre line that recombines with approximately 100% efficiency had no effect on proliferation or radial migration of astrocytes, but completely blocked radial migration of endothelial cells, strongly supporting the hypoxic astrocyte model. Using additional Cre driver lines, we found evidence for essential and partially redundant actions of retina-derived (paracrine) and astrocyte-derived (autocrine) VEGF in controlling astrocyte proliferation and migration. We also extended previous studies by showing that HIF-1α in retinal neurons and HIF-2α in Müller glia play distinct roles in retinal vascular development and disease, adding to a growing body of data that point to the specialization of these 2 hypoxia-sensing transcription factors.

Authors

Amir Rattner, John Williams, Jeremy Nathans

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Figure 3

Loss of intraretinal but not surface vasculature with loss of intraretinal VEGF.

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Loss of intraretinal but not surface vasculature with loss of intraretin...
(A–D) Vascular density in α-Cre Vegffl/fl retinas at P30. (A) Quantification of vascular density in the center (blue; little or no Cre-mediated recombination) and periphery (red; intraretinal Cre-mediated recombination) for each of the 3 vascular layers (vitreal surface, IPL, and OPL) in α-Cre Vegffl/fl retinas. In A, each symbol is the quantification from one 500-μm × 500-μm area; bars represent the mean ± SD and Q values were calculated with a 2-tailed unequal variance t test adjusted for multiple comparisons using the method of Benjamini and Hochberg (52). (B–D) GSL-stained vasculature at low magnification (B) and at intermediate magnification (C) and (D), corresponding, respectively, to the lower and upper labeled squares in B. The GSL signal is color coded by depth with the vitreal surface in blue, the IPL in green, and the OPL in red. Scale bars: 1 mm (B)and 200 μm (D). (E–H) Surface astrocyte density in α-Cre Vegffl/fl retinas at P30. E shows the quantification of the density of astrocyte processes (GFAP immunostaining) in the periphery of a WT retina (green) and in the center (blue) and periphery (red) of α-Cre Vegffl/fl retinas. Each symbol is the quantification from one 200-μm × 200-μm area; bars represent the mean ± SD, and Q values were calculated with a 2-tailed unequal variance t test adjusted for multiple comparisons using the method of Benjamini and Hochberg (52). Examples of the vitreal surface of WT peripheral retina and α-Cre Vegffl/fl central and peripheral retina following GFAP and GSL staining are shown at high magnification in F–H (scale bar: 50 μm). Images in G and H correspond to the labeled squares in C and D, respectively.