![Heme causes endothelial cell barrier disruption. (A) Heme entry into HLMVECs was quantified using the Hemin assay kit (Abcam; ab65332). HLMVECs treated with heme (50 μM, 15 minutes) had significantly increased intracellular heme content. (B) HLMVECs were seeded in ECIS plates, and after resistance measurements attained a stable level, heme was added to the media. Readings were recorded every minute after heme treatment and plotted as normalized cell index. (C) Rate of decline in resistance, reflective of barrier disruption, was calculated as slope using xCELLigence software. (D) Heme affects tight junction proteins. HLMVECs were treated with heme (50 μM, 60 minutes), and cells were fixed, immunostained for zona occludens-1 (ZO-1, green), and imaged using a confocal microscope (original magnification ×63). Arrows indicate the cellular organization of ZO-1. (E) Similarly, HLMVECs were treated with heme for the given amount of time and imaged at 20× and quantified for ZO-1 levels using Image J software. Images were quantified using at least 5 random images. (F-J) HLMVECs were treated with heme (50 µM) for different time points, and cell lysate was prepared in RIPA buffer with protease and phosphatase inhibitors. Endothelial junction proteins were detected using western blotting. Representative blots of ZO-1, claudin-1 [CLDN-1], CLDN-5, and VE-cadherin are shown (F), quantified using BioRad software (G-J). (K-L) Protein levels were normalized to β-actin. Stress fiber formation was seen after heme exposure. p22, the EF-hand protein required for actin polymerization, was found significantly increased. (M) HLMVECs were treated with heme (50 µM, 60 minutes), and cells were fixed, stained for F-actin using phalloidin (green), and imaged using a fluorescence microscope (original magnification ×63). Yellow arrows indicate apical stress fibers, and red arrows indicate stress fibers. Experiments were repeated at least 3 times. Values are mean ± standard error of the mean. *P < .05 compared with control. RFU, relative fluorescence unit.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/136/6/10.1182_blood.2019003986/3/bloodbld2019003986f1.png?Expires=1727816834&Signature=G3xmdgk~npIZp4Rw1XlscVXfrx1YoBAiGHwphO6ohppcu2-H0xZ-Fe8oVMBHEm~Ypv5KS8MBmBTm~8ToVv8ywPPRkgabiMkpTi2vN3q40Dq7k8oUJmJLopshV0JROFGYJNrzYDVNooG8OoW5xygmbyqxXDExaQvf9DCHjxekp3QQn3cf4~72lpkVj6ce3cIDQStENn72-JiqJbUDDGl5BKeF8TUREhDXJyO9fz9c5U0SYreqzugx36zRMg4OvfWEYOcrlcnW4OW4F3FysI6m31aWelZPi6T0hoJZQLTJmTW3EH6OgE8H67dtpb2WRXsfInsHHHGFRH87qUyfsn7hZw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Heme causes endothelial cell barrier disruption. (A) Heme entry into HLMVECs was quantified using the Hemin assay kit (Abcam; ab65332). HLMVECs treated with heme (50 μM, 15 minutes) had significantly increased intracellular heme content. (B) HLMVECs were seeded in ECIS plates, and after resistance measurements attained a stable level, heme was added to the media. Readings were recorded every minute after heme treatment and plotted as normalized cell index. (C) Rate of decline in resistance, reflective of barrier disruption, was calculated as slope using xCELLigence software. (D) Heme affects tight junction proteins. HLMVECs were treated with heme (50 μM, 60 minutes), and cells were fixed, immunostained for zona occludens-1 (ZO-1, green), and imaged using a confocal microscope (original magnification ×63). Arrows indicate the cellular organization of ZO-1. (E) Similarly, HLMVECs were treated with heme for the given amount of time and imaged at 20× and quantified for ZO-1 levels using Image J software. Images were quantified using at least 5 random images. (F-J) HLMVECs were treated with heme (50 µM) for different time points, and cell lysate was prepared in RIPA buffer with protease and phosphatase inhibitors. Endothelial junction proteins were detected using western blotting. Representative blots of ZO-1, claudin-1 [CLDN-1], CLDN-5, and VE-cadherin are shown (F), quantified using BioRad software (G-J). (K-L) Protein levels were normalized to β-actin. Stress fiber formation was seen after heme exposure. p22, the EF-hand protein required for actin polymerization, was found significantly increased. (M) HLMVECs were treated with heme (50 µM, 60 minutes), and cells were fixed, stained for F-actin using phalloidin (green), and imaged using a fluorescence microscope (original magnification ×63). Yellow arrows indicate apical stress fibers, and red arrows indicate stress fibers. Experiments were repeated at least 3 times. Values are mean ± standard error of the mean. *P < .05 compared with control. RFU, relative fluorescence unit.