With regard to angiogenesis, both the WT and
Emp2 KO animals showed an upregulation in a number of genes functionally associated with this process, including members of the
Hif and
Vegf families (
Fig. 5B). To characterize the expression of
Hif and
Vegf-A in the development of OIR, their levels were assessed by in situ hybridization, qRT-PCR, or immunohistochemistry. As expected, compared with age-matched normoxic controls,
Hif and
Vegfa mRNA abundance increased in all neuronal cell layers of the retina, including the retinal ganglion cell (RGC), inner nuclear layer (INL), and outer nuclear layer (ONL) in retinas of WT animals with OIR at P12 and P17 (
Figs. 6 and
7). The transcription factor Hif is upstream of VEGF signaling, with Hif upregulation beginning rapidly after exposure to hypoxia.
42–44 Hif expression in our OIR model peaks at P12 after removal of mice from hyperoxia (75% oxygen) and placement into “relative hypoxia” (21% oxygen), with normalization of Hif levels by P17 (
Fig. 6). VEGF expression peaks with maximal neovascular disease at P17 (
Fig. 7), both later and longer after initial “hypoxia” exposure, consistent with temporal expression patterns for transcriptional regulation.
42–44 Interestingly, in the P12 OIR mice,
Hif mRNA expression within the retinal neuronal layers was significantly higher in WT animals than in
Emp2 KO animals, as assessed by RNA in situ hybridization (
Fig. 6A;
P = 0.0006) and validated by qRT-PCR (
Fig. 6B;
P = 0.01). In the P12 and P17 OIR mice,
Vegfa mRNA expression within the RGC as well as the INLs and ONLs was significantly higher in WT animals than in the
Emp2 KO animals with OIR (
Fig. 7A;
P = 0.02 at P12;
P = 0.001 at P17). To validate these effects, protein abundance of
Vegf-A was confirmed by immunohistochemistry staining. P12 and P17
Emp2 KO animals showed decreased
Vegf-A protein expression within the RGC and INL layers of retinas with OIR compared with WT animals with OIR (
Fig. 7B;
P = 0.0002 at P12 and
P = 0.006 at P17). In comparison, age-matched animals from both groups maintained in normoxia showed extremely low mRNA levels of HIF and VEGFA.