Hypoxia-induced phosphorylation of astrocytic Cx43 is associated with misdirected angiogenesis in oxygen induced retinopathy

Abstract

Purpose : Hypoxia-induced degeneration of astrocytes contributes to the misdirected angiogenesis in oxygen-induced retinopathy (OIR). We studied the role of Cx43, the connexin isoform that forms gap junctions and hemichannels in astrocytes, in this degeneration and the associated pathology in OIR. To gain mechanistic insight, we examined changes in Cx43 phosphorylation, a key regulatory event known to affect Cx43 channel function, that occur prior to astrocyte loss. Finally, we explored whether preventing hypoxia-induced Cx43 phosphorylation increases astrocyte density and promotes reparative angiogenesis in OIR.

Methods : Mouse litters were exposed to 75% oxygen from postnatal day (p) 7 to p12 and then returned to room air. Cx43 was conditionally deleted from astrocytes using Cre-LoxP recombination (KO mice). Cx43 phosphorylation during relative hypoxia was examined with Western blot using phospho-specific antibodies. Kinase inhibitors as well as site-specific Cx43 phosphorylation-deficient mice (KI mice) were used to inhibit phosphorylation of Cx43. The degrees of vasobliteration (VO) and neovascularization (NV), and the astrocyte density were quantified using immunohistochemistry on retinal wholemounts from wild type (WT), KO and KI mice.

Results : Deletion of Cx43 markedly improved astrocyte density in OIR by p14 (WT, 257.8±14.5 cells/mm² vs KO, 471±37.7 cells/mm²; n=5, p=0.005), accelerated retinal revascularization (62.5±1.6% decrease in VO in KO mice compared to WT; n=8 mice, p<0.001) and reduced neovascularization (63±3.2% decrease in NV in KO mice compared to WT; n=8 mice, p<0.001). A 3-fold increase in Cx43 phosphorylation at CK1δ sites was detected within 6 hours of exposure to hypoxia (n=6, p=0.001). Retinae from mice injected with CK1δ inhibitors or KI mice that express Cx43 with CK1δ-site serine to alanine mutations exhibited enhanced directed angiogenesis (decreases of 41±2% in VO and 40±5% in NV in KI compared to WT; n=8, p<0.001) and elevated astrocyte density.

Conclusions : Phosphorylation of Cx43 by CK1δ occurs early in the hypoxia phase of OIR and facilitates astrocyte degeneration leading to pathologic angiogenesis. Targeting Cx43 channels or their phosphorylation are viable options for the treatment of ischemic retinopathies.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.