Abstract
Purpose :
The involvement of heme metabolism in pathological angiogenesis, a key hallmark of neovascular eye diseases like proliferative diabetic retinopathy and wet age-related macular degeneration (AMD) remains unclear. We previously identified the mitochondrial heme biosynthesis enzyme, ferrochelatase (FECH), as a mediator of angiogenesis. Inhibition or knockdown of FECH decreases key angiogenic properties of ocular endothelial cells in vitro and reduces ocular neovascularization in vivo without toxicity. FECH is overexpressed in human patient samples of wet AMD and in animal models of ocular neovascularization. One of the known downstream effects of FECH blockade is depletion of heme containing proteins, including complex IV (COX IV) of the electron transport chain (ETC). Hence, the goal of our study was to elucidate if FECH inhibition alters mitochondrial function of ocular endothelial cells via decreasing COX IV enzyme action.
Methods :
We measured oxygen consumption rate (OCR) of primary human retinal microvascular endothelial cells (HRECs) and a Rhesus choroidal endothelial cell line (RF/6A) using the Seahorse extracellular flux analyzer after FECH blockade. Similarly, we also measured extracellular acidification rate (ECAR) and determined glycolytic capacity. We immunoblotted proteins of the ETC and measured activity of COX IV by ELISA after FECH inhibition. Additionally, we rescued effects of FECH depletion by direct supplementation of heme to cells and measured COX IV function.
Results :
Key parameters of oxidative phosphorylation including maximal respiration, ATP production, spare respiratory capacity and proton leak decreased significantly after FECH inhibition in both endothelial cell types. FECH blockade also reduced glycolytic capacity and glycolytic reserve, as seen by a decrease in ECAR. Genetic and chemical inhibition of FECH reduced COX IV expression and activity by nearly 50%. This reduction was partially rescued after the cells were supplemented with heme.
Conclusions :
Our results confirm our hypothesis that FECH inhibition modulates endothelial mitochondrial physiology. Together, our findings suggest a novel mechanism of ocular angiogenesis, linking heme metabolism, mitochondrial function, and angiogenesis. Further studies to understand the hemylation patterns of COX IV after FECH mediated heme depletion are currently underway.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.