Abstract
Purpose :
Mutations in the heme transporter FLVCR2 cause Fowler syndrome, a prenatal lethal disease characterized by proliferative vasculopathy leading to glomeruloid bodies in the brain and retina. We hypothesize that intracellular heme content represents a completely novel regulator of angiogenesis regulating endothelial tip to stalk differentiation.
Methods :
Primary culture of retinal endothelial cells (BREC) were used to test the effect of siRNA to FLVCR2. Cell proliferation was assessed by EdU incorporation or ki67 immunostaining. Tube formation was determined by Matrigel assay in vitro and in retinal explants ex vivo. Heme synthesis was inhibited by using succinylacetone (SA) and induced by 5-Aminolevulinic acid (5ALA). VEGF signaling and cleaved NOTCH product were determined by Western blot. Endothelial-specific knockout of FLVCR2 was achieved by Cdh5-Cre recombination in mice.
Results :
Knockdown of FLVCR2 in BREC led to increased proliferation and tube formation, coincident with a 50% decrease in intracellular heme content. Inhibiting heme biosynthesis with SA was able to mimic the effects of FLVCR2 knockdown. Conversely, adding back the heme precursor 5ALA or membrane soluble hemin reversed the effects of FLVCR2 knockdown and SA, respectively. Decreasing intracellular heme levels by either FLVCR2 knockdown or SA did not alter VEGF signaling, nor did it alter mitochondrial function as measured by Seahorse. NOTCH signaling pathway was increased by FLVCR2 knockdown and blocking NOTCH cleavage with gamma-secretase inhibitor DAPT was able to completely prevent the increase in cell proliferation and tube formation. In retinal explants decreased intracellular heme levels promoted increased sprout length but only when provided after tip cell formation and initiation of angiogenesis. In a similar manner, inducing endothelial-specific FLVCR2 knockout at post-natal day 7 led to the formation of glomeruloid structures and hypoxia in the superficial capillary plexus of the retina. Additionally, systemic administration of SA lead to an underdevelopment of the deep capillary plexus in the retinas of wild-type mice.
Conclusions :
Together, these results suggest that low levels of intracellular heme in retinal endothelial cells promote the stalk cell phenotype increasing proliferation and tube formation and reveal heme as a novel regulator of retinal angiogenesis.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.