%0 Journal Article
%T Heme accumulation in endothelial cells impairs angiogenesis by triggering paraptosis
%A Deborah Chiabrando
%A Emanuela Tolosano
%A Federico Mussano
%A Fiorella Altruda
%A Francesca Vinchi
%A Giada Ingoglia
%A Giorgio Roberto Merlo
%A Lorenzo Silengo
%A Luca Munaron
%A Sara Petrillo
%A Stefano Carossa
%A Tullio Genova
%A Veronica Fiorito
%J Archive of "Cell Death and Differentiation".
%D 2018
%R 10.1038/s41418-017-0001-7
%X 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£¿¡À£¿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 ¦Ä-aminolevulinic acid (ALA) 5£¿mM for 2¨C4£¿h, are shown in k. Values are expressed as pmol heme/mg protein. Data represent mean£¿¡À£¿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£¿¡À£¿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£¿¡À£¿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£¿¡À£¿SEM, n£¿=£¿3; *p£¿<£¿0.05. ALAS1 ¦Ä-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 ¦Ä-aminolevulinic acid; PI propidium iodid
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5864215/