Extended Data Fig. 10: Lysed RBC membranes form an endovascular sealant (Endo-seal).
From: Ischaemic endothelial necroptosis induces haemolysis and COVID-19 angiopathy
a, Schematic of RBC lysis and Endo-seal formation on dying endothelium. b, Intravital fluorescence time-lapse imaging depicting progressive RBC lysis (TER-119+H) localized at dying ECs (AnV/PS+; S1, S2, S3) in submucosal vessels during reperfusion (5 IR mice); Scale bar, 10 µm. c, Intravital fluorescence time-lapse images showing repetitive cycles of RBC elongation/fragmentation (arrows) lead to multilayers of Endo-seal formation on IR injured submucosal micro-vessel wall at the indicated time during reperfusion (4 mice); Scale bar, 10 µm. d. 3D-confocal/Imaris images of capillary segments showing progressive thickening of RBC Endo-seal (longitudinal view: left; zoomed cross-section views: middle and right) in IR-injured villi (4 mice); Scale bars, 20 and 5 µm. e, Quantification of COVID-19 patient tissue microvasculature containing Endo-seal (CD235+H) versus intact RBCs (CD235+L) (kidney, heart, and liver, n = 124 vessels/organ, 3 organs, 3 patients), f, Representative confocal and correlated H&E images of the same COVID-19 kidney vessel depicting lysed RBC-derived Endo-seal (CD235+H) without platelet thrombi and fibrin on dying endothelium (arrows), but not viable endothelium (arrowhead) lacking Endo-seal (4 organs/4 COVID-19 patients). Scale bars, 5 µm. g(i,ii), Representative IF/DIC images of lysed RBC-derived Endo-seal (CD235+H, arrows) in vessels from COVID-19 heart, kidney and liver tissues (27 organs/8 COVID-19 patients) (g-i); and ischaemic patient heart (AMI), brain (stroke) and colon tissues (5 heart, 1 brain, 3 colon tissues, >20 vessels/tissue) (g-ii). Scale bars, 20 µm. h, Representative airyscan and B-SEM images showing progression of RBC Endo-seal formation (TER-119+H) from thin to thick layers and eventual occlusion in mouse villus capillary segments, demonstrating first, the narrowing of TER-119+H-free lumen indicated by red arrows (middle panels), and second, the thickening of Endo-seal and finally occlusion (B-SEM: shaded in pink, blue arrows) on the damaged endothelium shaded in yellow in B-SEM (bottom panels) (4 IR mice). Scale bars, 10 µm (top panels) and 2 µm (bottom panels). Data represent mean ± SD. Statistical analysis: two-sided t-test (e).
