Heme accumulation in endothelial cells impairs angiogenesis by triggering paraptosis

a-h qRT-PCR analysis of FLVCR1a a, c, FLVCR1b b, d, ALAS1 e, g and HO1 f, h in HMECs a, b, e, f or HUVECs c, d, g, h in which the expression of FLVCR1a was downregulated using a specific shRNA 1a. Transcript abundance, normalized to 18s mRNA expression, is expressed as a fold increase over a calibrator sample. Data represent mean？±？SEM, n？=？5; *p？<？0.05, **p？<？0.01, ***p？<？0.001. i, k Heme content in HMECs i or HUVECs k in which the expression of FLVCR1a was downregulated using a specific shRNA. Intracellular heme levels in HUVECs cultured in basal condition or treated with δ-aminolevulinic acid (ALA) 5？mM for 2–4？h, are shown in k. Values are expressed as pmol heme/mg protein. Data represent mean？±？SEM, n？=？10; *p？<？0.05, **p？<？0.01, ***p？<？0.001, ****p？<？0.0001. j, l ROS amount measured by detection of H2DCFDA fluorescence in HMECs j or HUVECs l with or without FLVCR1a in basal condition or after treatment with ALA 5？mM for 15？h. Values are expressed as FIU/mg protein. Data represent mean？±？SEM, n？=？4; *p？<？0.05. m, o Cell viability assessed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of HMECs m or HUVECs o in which the expression of FLVCR1a was downregulated using a specific shRNA. Values are expressed as percentage increase at 24, 48？h compared with T0 (time zero). Data represent mean？±？SEM, n？=？8 (HMECs), n？=？3 (HUVECs); ***p？<？0.001, ****p？<？0.0001. n, p Flow cytometry assay performed on HMECs n or HUVECs p with or without FLVCR1a, after incubation with Annexin V-FITC antibody. Values are expressed as percentage of Annexin V high/PI low cells. Data represent mean？±？SEM, n？=？3; *p？<？0.05. ALAS1 δ-aminolevulinate synthase-1; HO1 heme oxygenase-1; RQ relative quantification; qRT-PCR quantitative real-time PCR; ROS reactive oxygen species; shRNA short hairpin RNA; ALA δ-aminolevulinic acid; PI propidium iodid