Ferrochelatase is a novel mediator of ocular angiogenesis

Abstract

Purpose: Prevention of neovascularization is a key strategy for the treatment of wet age-related macular degeneration (AMD). Current antiangiogenic biologics act by blockade of vascular endothelial growth factor (VEGF) signaling at the VEGF ligand-receptor level and more than 30% patients are refractive to these drugs. Hence there is a critical need to identify novel angiogenesis mediators that can be targeted to complement anti-VEGF approaches. The purpose of the present study was to find novel protein targets that can be exploited to inhibit angiogenesis. We have identified ferrochelatase (FECH) as a novel and critical angiogenesis mediator. FECH is the enzyme responsible for protoporphyrin IX metallation, the rate limiting step of heme biosynthesis.

Methods: Photoaffinity chromatography was used to identify the interacting partners of a natural antiangiogenic molecule, cremastranone. The pulled down proteins were identified by mass spectrometry and confirmed by immunoblot and competition experiments. The role of FECH in angiogenesis was evaluated in vitro by modulating its levels by siRNA in human retinal microvascular endothelial cells (HRECs) and monitoring the angiogenic properties of HRECs such as proliferation, migration, and Matrigel tube formation assays. The importance of FECH for angiogenesis was confirmed in vivo using the laser-induced choroidal neovascularization (L-CNV) mouse model. The expression of key proangiogenic molecules was monitored after FECH knockdown.

Results: FECH was pulled down as an interacting protein partner of cremastranone and confirmed as a target of this antiangiogenic compound by immunoblot and competition off the affinity beads with a cremastranone analog. Cremastranone inhibited FECH activity in HRECs at sub-micromolar concentration. Knockdown of FECH in HRECs resulted in complete inhibition of angiogenesis in vitro, as assessed by HREC proliferation, migration, and tube formation. However, knockdown did not induce apoptosis in these cells and had minimal effect on non-endothelial cell lines. Knockdown of FECH in HRECs resulted in decreased protein levels of key angiogenic factors HIF-1α, eNOS and hemoxygenase-1. In the L-CNV model, siRNA against FECH markedly inhibited choroidal neovascularization.

Conclusions: FECH plays a crucial, previously undocumented, role in angiogenesis. Inhibition of ferrochelatase activity might be exploited to inhibit the ocular neovascularization that underlies wet AMD.