Abstract
Purpose :
Non-apoptotic induction of endothelial caspase-9 by retinal vein occlusion (RVO) sets off a signaling pathway causing edema, BRB breakdown, and eventual neuronal loss, however the exact mechanism of this pathway is still unknown. Previous studies by our lab have shown that knocking out endothelial caspase-9 diminishes the amount of edema and neuronal damage after RVO, suggesting that the endothelial caspase-9 is motivating the signaling pathway. Since this large induction of caspase-9 seems to be non-apoptotic which is atypical of the normal function, we are determining if the upstream activation of caspase-9 by Apaf-1 is the driving force behind the non-apoptotic signaling. These experiments will be carried out using inducible endothelial cell Apaf-1 knock out mice (Apaf-1 iECKO) and WT littermates who are injured with retinal vein occlusions.
Methods :
2-month-old Apaf-1 iECKO and WT littermate mice will undergo the RVO procedure previously described in Avrutsky, et al. Live imaging readouts will be used to track the pathology of the animals and compare to previous data from the caspase-9 iECKO mice. Immunohistochemistry and western blotting will be used to determine the presence of a known downstream target caspase-7 and to track the alteration in other proteins relevant to edema, BRB breakdown, and neuronal loss between injured vs. non-injured mice as well as the Apaf-1 iECKO vs. WT.
Results :
This model has previously shown that the deletion of caspase-9 protects from edema and neuronal injury after RVO. Co-staining with TUNEL (marker for cell death) shows that endothelial cells aren’t dying but neurons are. We know that this activation of caspase-9 within endothelial cells is non-apoptotic and will explore the activation of endothelial caspase-9 to determine why it is not carrying out its normal death signaling in the endothelial cells themselves but instead setting off a signaling cascade leading to neuronal death.
Conclusions :
Endothelial caspase-9 sets off a signaling cascade leading to neuronal death and by eliminating the induction of endothelial caspase-9, we are able to prevent edema and BRB breakdown while preserving the neurons. The activation of caspase-9 specifically in endothelial cells seems to be different than its canonical function. Apaf-1 activation may be altered in this system and can be driving how caspase-9 is behaving in different cell types.
This is a 2021 ARVO Annual Meeting abstract.