Hemoglobin oxidation generates globin-derived peptides in atherosclerotic lesions and intraventricular hemorrhage of the brain, provoking endothelial dysfunction

Niké Posta,Éva Csősz,Melinda Oros,Dávid Pethő,László Potor,Gergő Kalló,Zoltán Hendrik,Katalin Éva Sikura,Gábor Méhes,Csaba Tóth,József Posta,György Balla,József Balla

doi:10.1038/s41374-020-0403-x

Abstract

The lysis of red blood cells was shown to occur in human ruptured atherosclerotic lesions and intraventricular hemorrhage (IVH) of the brain. Liberated cell-free hemoglobin was found to undergo oxidation in both pathologies. We hypothesize that hemoglobin-derived peptides are generated during hemoglobin oxidation both in complicated atherosclerotic lesions and IVH of the brain, triggering endothelial cell dysfunction. Oxidized hemoglobin and its products were followed with spectrophotometry, LC–MS/MS analysis and detection of the cross-linking of globin chains in complicated atherosclerotic lesions of the human carotid artery and the hemorrhaged cerebrospinal liquid of preterm infants. The vascular pathophysiologic role of oxidized hemoglobin and the resultant peptides was assessed by measuring endothelial integrity, the activation of endothelial cells and the induction of proinflammatory genes. Peptide fragments of hemoglobin (VNVDEVGGEALGRLLVVYPWTQR, LLVVYPWTQR, MFLSFPTTK, VGAHAGEYGAELERMFLSFPTTK, and FLASVSTVLTSKYR) were identified in ruptured atherosclerotic lesions and in IVH of the human brain. Fragments resulting from the oxidation of hemoglobin were accompanied by the accumulation of ferryl hemoglobin. Similar to complicated atherosclerotic lesions of the human carotid artery, a high level of oxidized and cross-linked hemoglobin was observed in the cerebrospinal fluid after IVH. Haptoglobin inhibited hemoglobin fragmentation provoked by peroxide. The resultant peptides failed to bind haptoglobin or albumin. Peptides derived from hemoglobin oxidation and ferryl hemoglobin induced intercellular gap formation, decreased junctional resistance in the endothelium, and enhanced monocyte adhesion to endothelial cells. Enhanced expression of TNF and the activation of NLRP3 and CASP1 followed by the increased generation of IL-1β and nuclear translocation of the NF-κβ transcription factor occurred in response to hemoglobin-derived peptides, and ferryl hemoglobin in endothelium was upregulated in both pathologies. We conclude that the oxidation of hemoglobin in complicated atherosclerotic lesions and intraventricular hemorrhage of the brain generates peptide fragments and ferryl hemoglobin with the potential to trigger endothelial cell dysfunction.

Highlights

The lysis of red blood cells occurs in several pathologic conditions [1]
We previously described the oxidation of hemoglobin and the formation of ferryl hemoglobin in human atherosclerotic lesions [9]
We examined the pathophysiologic role of hemoglobin oxidation and the fate of hemoglobin in these pathologies

Summary

Introduction

The lysis of red blood cells occurs in several pathologic conditions [1]. Cell-free hemoglobin α2β2 tetramers dissociate into α, β dimers, which are oxidized into methemoglobin (Fe3+) [2, 3]. Exposure of extracellular hemoglobin to reactive oxygen species, e.g., hydrogen peroxide (H2O2), or lipid hydroperoxide can lead to the formation of short-lived ferryl (Fe4+ = O2−) hemoglobin [4, 5]. Ferryl hemoglobin has the propensity to form free radicals in the alpha and beta chain of the globin [6,7,8]. Globin–globin cross-links are formed by globin-centered radicals, resulting in hemoglobin dimers, tetramers and multimers, and heme (Fe2+). We refer to these globin-modified molecules as ferryl hemoglobin

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