Red Blood Cells and Hemoglobin in Human Atherosclerosis and Related Arterial Diseases

Figures

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  Figure 1. Macroscopic view of hemorrhages in human arterial lesions: (A) involvement of RBCs in the initial stages of human atherosclerotic lesions ((A1) aortic fatty streaks in human aorta and their relationship to intercostal ostia; (A2) fibro-atheroma with limited subintimal bleeding; (A3) more advanced lesions with numerous small hemorrhages and intimal breaches); (B) 0stial hematoma ((B1) ostial luminal view; (B2) outside collateral view; (B3) longitudinal trans-ostial section showing the local diffusion of the hematoma; and (B4) whole-mount immunostaining of Glycophorin A in peri-ostial clarified aortic tissue showing the hematoma diffusion from the ostia (*)); and (C) ILT in AAA and aortic valve; (C1) the multilayered intraluminal thrombus (ILT) of human AAA, where the most luminal layer (red) is extremely rich in RBCs and the subjacent layers are yellow/brown, demonstrating the release of heme and ferrous iron, and the metabolism of hemoglobin into bilirubin; (C2) aortic view of a calcified aortic valve, with peri-calcification hemorrhages (red), where the macro-crystallized calcifications protrude from the fibrosa; and (C3) view of the ventricularis of the same valve where the…

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  Figure 2. Histological views of tissue hemolysis in human aortic plaques and valves: (A) atherosclerosis ((A1) Perl’s + DAB staining of redox-active Fe++ in the shoulder of an atherosclerotic plaque; (A2) ORO lipid staining, colocalizing with Fe++ on a serial section; (A3) high magnification of foam SMCs by Prussian blue precipitation, showing the presence of Fe+++, potentially linked to ferritin (redox-inactive); and (A4) autofluorescent (550-nm wavelength, red) ceroid rings in an atherosclerotic plaque); and (B) calcified aortic valves ((B1) a crown of ferrous iron around calcification in an aortic valve, suggesting a compliant mismatch between the solid calcification and the soft elastic valve tissue; and (B2) red autofluorescence (550-nm wavelength) directly associated with aortic valve calcification).

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  Figure 3. Phagocytosis of senescent RBCs by cultured human SMCs: (A) SMCs exposed to senescent sRBCs ((A1) acquisition of CD68, the phagolysosome functional marker of phagocytosis activity by cultured SMCs, usually used as a marker of macrophages, but not lineage-specific; (A2) acquisition of intracellular Fe++ staining; (A3) acquisition of lipid staining from sRBC membranes within SMCs; (A4) early phagocytosis of intact sRBCs, stained by DAB alone (pseudo-peroxidase activity); and (A5) electron microscopic view of efferocytosis of sRBCs by cultured human SMCs with the presence of extracellular RBC skeletons (*), containing hyperdense bodies, potentially iron and extracellular release of numerous micro-vesicles and exosomes by the sRBC-phagocytic SMCs); and (B) valvular Interstitial Cell (VIC) ((B1) efferocytosis of sRBCs (red) by human VIC).

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  Figure 4. RBC-dependent oxidative stress in AAA: (A) macroscopic luminal view of the thrombus, with a predominant RBC fresh clotting at the interface with circulating blood; (B) diaminobenzidin (DAB) staining of RBC in the most luminal layer; (C) autofluorescent ceroids (550 nm), the wavelength of Hb; (D) oil red O staining of lipids (orange color); and (E) Perl’s blue staining of Fe+++, the storage redox inactive form of iron, bound to ferritin and transported by transferrin (recycling).