Abstract
Purpose: :
Hypoxia-inducible factors (HIFs) are central members of the molecular oxygen sensing pathway, and their role in response to hypoxia is well known. The retina of the newborn mouse is virtually avascular lacking especially the two inner vascular plexi that only form within the first two weeks after birth. This suggests reduced tissue oxygenation at this time requiring an appropriate molecular response. Indeed, HIF-1α levels are high in the young mouse retina but decrease as the retinal vasculature develops. Here, we tested the involvement of HIF-1α in vascular development in the mouse retina.
Methods: :
Conditional HIF-1α knockdowns were generated by breeding HIF1αflox/flox mice to mice expressing cre recombinase under control of the alpha element of the Pax6 promoter. This leads to a deletion of HIF-1α genomic DNA and thus to HIF-1α knockdown in cells of the peripheral but not the central retina. Retinal morphology was observed in mice by light microscopy. Development of retinal blood vessels was analyzed using IHC on retinal flat mounts. Gene expression was assessed by semiquantitative real-time PCR and Western blotting.
Results: :
HIF-1α knockdown mice had a 50% reduced global retinal expression of HIF-1α at post natal day (PND) 21. The peripheral retinal vasculature of HIF1α knockdown mice was generally underdeveloped and less organized and the development of the inner vascular plexi was disturbed as compared to control mice. The phenotype was most pronounced at PND10 and persisted into adulthood. In contrast, retinal morphology was largely unaffected. Increased levels of GFAP mRNA suggested retinal stress at PND21. Surprisingly, levels of pan-VEGF mRNA were increased by 50% at PND21 despite the lower expression of HIF-1α. Expression of Egln1 and Glut1, other HIF-1α target genes, was largely unaffected
Conclusions: :
The underdeveloped retinal vasculature in the HIF-1α knockdown mice strongly suggests a prominent role of HIF-1α in retinal angiogenesis and especially in the formation of the deep retinal vessels. In an attempt to form retinal blood vessels, retinal cells may increase VEGF expression in a HIF-1α independent manner or in cells not affected in by the HIF-1α gene knockdown.
Keywords: hypoxia • development • vascular endothelial growth factor