Hypoxia in OIR mice decreases with ongoing retinal development and cell differentiation.
19 Endoplasmic reticulum stress has also been found to be a part of normal retinal and lens embryonic development.
20,21 In support of these findings, the results obtained in our laboratory suggest that the activation of the UPR occurs in rodent retinas during the first 2 weeks of life (data not shown). Therefore, given that the mouse retina experiences modulation of many genes during development
22 and that programmed cell death naturally occurs in the developing vertebrate retina,
23 we decided to dissect the role of hypoxia-driven alteration of gene expression in ATF4-deficient retinas from the effect of genetic manipulation with ATF4 in developing retinas and to analyze the UPR-associated gene expression in the ATF4
+/− mice. The list of studied genes includes the UPR-associated
ATF6,
CHOP (DDIT3 or DNA damage–inducible factor 3),
Bip (
Grp78),
eIF2α,
Xbp1,
Bax (BCL2-associated X protein), and
Hif1α, all of which were shown to be modulated in DR.
4,13,24 A major role of phosphatidylinositide kinases/v-akt murine thymoma viral oncogene (protein kinase B) (Pi3K/AKT) signaling has been described in the induction of angiogenesis and the prolonging of cellular survival in the proliferative stage of DR
25 and therefore was also tested in this study. Our selection of the mitogen-activated protein kinase 3 (or ERK1) (MAPK3) was based on ERK1 involvement in VEGF release in diabetic retinas.
26,27 For this reason, we studied the
Mapk3 gene, along with
Vegfα,
Flt1 (VEGF receptor 1), and
Pik3r1 (phosphoinositide-3-kinase, regulatory subunit 1).
Tgfβ1 (transforming growth factor β1) expression and secretion have been shown to increase in diabetic retinas and were thus also of interest to us.
Tgfβ1 was also proposed together with VEGF and IGF1 to stimulate residual vessel proliferation.
28 In addition, we selected this factor based on the proposed link between ATF4 ablation and downregulation of TGFb expression in neuronal cells.
29