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, other caspases, and proteins known to be associated with other cell death pathways. TUNEL staining will be used to measure the amount of cell death present after injury in knockout and wild type littermates.
Results :
Apaf-1 iECKO mice show less edema after injury than Apaf-1 WT littermates measured using total retinal thickness. They also do not show the same level of thinning 8 days post injury suggesting that there is more neuronal death in the WT animals than the KOs. Apaf-1 iECKOs have lower expression of caspase-9 and direct target caspase-7 as well as less TUNEL staining, indicating less neuronal death.
Conclusions :
The activation of caspase-9 specifically in endothelial cells seems to be different than its canonical function however we see that Apaf-1 iECKO is protective in many of the same ways that caspase-9 iECKO is. This suggests that although endothelial caspase-9 is not leading to cell death within the endothelial cells, it is still being activated by Apaf-1. This suggests that the activation of caspase-9 is not driving the non-apoptotic function in endothelial cells and may instead be due to the downstream signaling.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.