Astrocyte-derived vascular endothelial growth factor stabilizes vessels in the developing retinal vasculature

Abstract

Vascular endothelial growth factor (VEGF) plays a critical role in normal development as well as retinal vasculature disease. During retinal vascularization, VEGF is most strongly expressed by not yet vascularized retinal astrocytes, but also by retinal astrocytes within the developing vascular plexus, suggesting a role for retinal astrocyte-derived VEGF in angiogenesis and vessel network maturation. To test the role of astrocyte-derived VEGF, we used Cre-lox technology in mice to delete VEGF in retinal astrocytes during development. Surprisingly, this only had a minor impact on retinal vasculature development, with only small decreases in plexus spreading, endothelial cell proliferation and survival observed. In contrast, astrocyte VEGF deletion had more pronounced effects on hyperoxia-induced vaso-obliteration and led to the regression of smooth muscle cell-coated radial arteries and veins, which are usually resistant to the vessel-collapsing effects of hyperoxia. These results suggest that VEGF production from retinal astrocytes is relatively dispensable during development, but performs vessel stabilizing functions in the retinal vasculature and might be relevant for retinopathy of prematurity in humans.

Figure 1. Astrocyte specific deletion of VEGF.

(A, B) In situ hybridisation showed Vegf mRNA expression in a retinal cross-section (A) and a retinal whole mount (B) at P5. (A) Vegf mRNA was most strongly expressed in retinal astrocytes (arrowhead), and weakly in retinal ganglion cells and the inner nuclear layer (arrows). (B) Retinal astrocytes expressed VEGF strongest distally to the edge of growing vascular plexus (white arrowheads), at intermediate levels around veins (v) and weakest along arteries (a). (C) Transgenic mice expressing Cre recombinase in retinal astrocytes (Gfap-Cre mice) showed in a lacZ reporter strain recombination activity (black X-gal stain in C) in retinal astrocytes proximally and distally to the growing edge of the vascular plexus (arrowheads in C). (D, E) Cre recombination in Vegf ^c/c mice, crossed with Gfap-Cre mice, was demonstrated by PCR across the deleted region in genomic DNA from retinas in Cre positive (mt) and Cre negative (wt) animals (D). PCR on tail DNA identified Cre negative and positive animals (E). (F) PCR on cDNA from retinal mRNA showed unchanged VEGF isoform ratios in control and mutant animals. Astrocyte deletion of VEGF led to subtle abnormalities in the P5 retinal vasculature (stained with an anti-claudin 5 antibody G–J) in Cre positive animals (H) compared to wild type litter mates (G). In contrast, deletion of HIF1α in retinal astrocytes caused no such abnormalities (I–J). Scale bars are 100µm in A, 200µm in B, C and 1000µm in G.

Figure 2. Effects of astrocyte-derived VEGF on retinal vascular development.

Immunohistochemistry, visualizing endothelial cells (claudin 5, green in A, B, C, E, G) and retinal astrocytes (GFAP, red in A, B), shows that angiogenic sprouting at the leading edge of the growing vascular plexus appears normal in control (A) and mutant (B) animals lacking astrocyte derived VEGF. (C, D) Measurement of the surface area of the retinal vasculature (C) showed significantly reduced spreading in Vegf ^c/c mutants at P5 but not at P10 and not in P5 Hif1a ^c/c mutants at P5. (E) AT P10 proliferation (anti-BrdU in red) occurs predominantly in veins and is reduced in animals lacking astrocyte-derived VEGF in comparison to littermate controls. (G–H) Endothelial cell survival near arteries was assessed by measuring the width of the capillary free zone (CFS, white arrows) and the number of artery side branches (black arrowheads). (H) In mutant animals CFS width was not affected but the number of side branches was reduced. Scale bars are 50µm in A, 1000µm in C and 200µm in G; * is p<0.05 and ** is p<0.01.

Figure 3. Astrocyte-derived VEGF protects vessels from hyperoxia.

After hyperoxia exposure from P7–12 immunohistochemistry was used to visualize vessels with isolectin B4 (green, A, B), collagen IV (green, D, E) and retinal astrocytes (GFAP, red, D, E). (A–C) Deletion of astrocyte specific VEGF increased the vaso-obliterated area and led to decreased survival of radial arteries and veins. In some instances this lead to non-perfused, hyperproliferating capillary beds in the periphery (arrowhead in B). (D, E) Retinal astrocyte survival was not affected at this age but in areas of capillary loss astrocytes re-aligned with nerve bundles (arrowheads D, E). Scale bars are 100µm in A and 100µm in D; ** is p<0.01.

Figure 4. Vessel degeneration after one day of hyperoxia (P7–8).

(A–C) In situ hybridization showed that Vegf mRNA was detectable within the vascular network (stained with anti-collagen IV, red) but strongly reduced around arteries. Immunohistochemistry with anti-collagen IV (red D–F, green G, H), anti-claudin 5 (green D–F) and active caspase 3 (red G, H) revealed dying vessels. Empty basement membrane sleeves (arrows D) and isolated claudin 5 positive clumps were indicative of a regressing capillary network and local narrowing of artery and vein profiles (arrowheads E, F) suggested blood flow reductions. (G–I) Astrocyte-specific VEGF deletion increased radial vessel degeneration and affected veins more strongly than arteries at this early time point. Scale bars are 200 µm in A and 50 µm in D–G; * is p<0.05 and *** is p<0.001.