Purpose: The activation of the unfolded protein response (UPR) and an increase in activating transcription factor 4 (ATF4) has been previously reported in the diabetic retina. Despite this, a direct link between ATF4 and the degree of proliferative retinopathy has not been demonstrated to date. Therefore, the objective of this study was to determine whether ATF4 deficiency could reduce neovascularization in mice with oxygen-induced retinopathy (OIR).
Methods: We induced OIR in C57BL/6, ATF4(+/-), and endoplasmic reticulum stress-activated indicator (ERAI) mice and used quantitative RT-PCR and Western blot analysis to evaluate relative gene and protein expression. Histology and microscopy were used to calculate the extent of neovascularization in flat-mounted retinas.
Results: Experimental data revealed Xbp1 splicing in the retinal ganglia cells, outer plexiform layer, inner nuclear layer, and outer nuclear layer and in pericytes of postdevelopment day 17 ERAI OIR mice, confirming the activation of IRE1 UPR signaling. In naive ATF4-deficient mice, we also observed an elevation in UPR-associated and vascular-associated gene expression (Bip, Atf6, Hif1a, Pik3/Akt, Flt1/Vegfa, and Tgfb1), which may have contributed to the alleviation of hypoxia-driven neovascularization in experimental ATF4(+/-) retinas. The OIR ATF4(+/-) retinas demonstrated reprogramming of the UPR seen at both the mRNA (Atf6 and Bip) and protein (pATF6 and peIf2α) levels, as well as a reduction in vascularization-associated gene expression (Flt1, Vegf1, Hif1, and Tgb1). These changes corresponded to the decline in the rate of neovascularization.
Conclusions: Our study validates ATF4 as a prospective therapeutic target to inhibit neovascularization in proliferative retinopathy.
Keywords: activating transcription factor 4; angiogenesis; oxygen-induced retinopathy; unfolded protein response; vascular endothelial growth factor.