Role of ferrochelatase in mitochondrial bioenergetics of ocular endothelial cells

Abstract

BackgroundAberrant angiogenesis drives the pathology of many diseases like atherosclerosis, diabetic retinopathy, rheumatoid arthritis and tumorigenesis. Regenerative angiogenesis has also been suggested as a form of therapy for treating ischemic coronary diseases that necessitate revascularization. We previously identified the heme biosynthesis enzyme, ferrochelatase (FECH), as a mediator of angiogenesis. Inhibition or knockdown of FECH significantly decreases key angiogenic properties of ocular endothelial cell types in vitro and reduces neovascularization in vivo without toxicity. FECH overexpression is seen in human patient samples of neovascular eye disease as well as in animal models of ocular neovascularization. However, the mechanistic role of FECH in angiogenesis has not yet been described. One of the known downstream effects of FECH blockade is depletion of heme and heme‐containing proteins. One particularly sensitive hemoprotein is mitochondrial complex IV (Cox IV) of the electron transport chain (ETC). Hence, we hypothesized that inhibition of FECH affects Cox IV expression and alters the mitochondrial function of ocular endothelial cells.MethodsWe determined the expression of Cox IV subunit 1 in primary human retinal microvascular endothelial cells (HRECs). We also studied the ETC and mitochondrial bioenergetics of ocular endothelial cells using SeaHorse extracellular flux analyses. We first characterized the cell energy phenotype of HRECs and the Rf/6A Rhesus choroidal endothelial cell line. We measured oxygen consumption rate and extracellular acidification rate after FECH inhibition or siRNA knockdown. Mitochondrial stress was induced using a serial injection of ETC inhibitors oligomycin, carbonyl cyanide‐4‐(trifluoromethoxy)‐phenylhydrazone (FCCP), rotenone, and antimycin A. Similarly, we also measured glycolytic capacity of cells after FECH inhibition and induced stress on cells by glucose starvation followed by stimulation of glycolysis with glucose injection and blockade of mitochondrial ATP production by oligomycin.ResultsWe observed a reduction in expression levels of Cox IV on knockdown of FECH in HRECs. Rf/6A cells are more glycolytic under stress, whereas HRECs have the propensity to meet energy demands from either mitochondrial respiration or glycolysis when stressed. Knockdown and chemical inhibition of FECH in HRECs and Rf/6A cells led to a dramatic decrease in maximal respiration, spare respiratory capacity, ATP production, and proton leak while lowering the coupling efficiency on induction of mitochondrial stress. Further, we also observed that glycolysis, glycolytic capacity, and glycolytic reserve were decreased on the reduction of FECH.ConclusionTogether, our findings characterize for the first time the mitochondrial energetics of ocular cell types that underlie neovascular diseases, and reveal a novel link between heme metabolism, mitochondrial function, and angiogenesis. Further studies to understand the mitochondrial morphology, membrane potential and production of mitochondrial reactive oxygen species in response to FECH modulation are currently underway.Support or Funding InformationNIH/NEI R01EY025641, Research to Prevent Blindness, Inc.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.