Endothelial Barrier Integrity Is Disrupted In Vitro by Heme and by Serum From Sickle Cell Disease Patients.

Vanessa Araujo Gomes Santaterra,Bidossessi Wilfried Hounkpe,Aderson Da Silva Araújo,Loredana Nilkenes Gomes Da Costa,Wouitchekpo Vincent Tonasse,Antonio Roberto Lucena-Araújo,Maiara Marx Luz Fiusa,Diego Garcia-Weber,Magnun Nueldo Nunes Dos Santos,Francine Chenou,Franciele De Lima,Fernando Ferreira Costa,Erich Vinicius De Paula,Ivanio Teixeira Borba-Junior,Jaime Millán,Igor De Farias Domingos,Marcos André Cavalcante Bezerra

doi:10.3389/fimmu.2020.535147

Abstract

Free extracellular heme has been shown to activate several compartments of innate immunity, acting as a danger-associated molecular pattern (DAMP) in hemolytic diseases. Although localized endothelial barrier (EB) disruption is an important part of inflammation that allows circulating leukocytes to reach inflamed tissues, non-localized/deregulated disruption of the EB can lead to widespread microvascular hyperpermeability and secondary tissue damage. In mouse models of sickle cell disease (SCD), EB disruption has been associated with the development of a form of acute lung injury that closely resembles acute chest syndrome (ACS), and that can be elicited by acute heme infusion. Here we explored the effect of heme on EB integrity using human endothelial cell monolayers, in experimental conditions that include elements that more closely resemble in vivo conditions. EB integrity was assessed by electric cell-substrate impedance sensing in the presence of varying concentrations of heme and sera from SCD patients or healthy volunteers. Heme caused a dose-dependent decrease of the electrical resistance of cell monolayers, consistent with EB disruption, which was confirmed by staining of junction protein VE-cadherin. In addition, sera from SCD patients, but not from healthy volunteers, were also capable to induce EB disruption. Interestingly, these effects were not associated with total heme levels in serum. However, when heme was added to sera from SCD patients, but not from healthy volunteers, EB disruption could be elicited, and this effect was associated with hemopexin serum levels. Together our in vitro studies provide additional support to the concept of heme as a DAMP in hemolytic conditions.

Highlights

Heme is a ubiquitous molecule present in almost all forms of life, that is normally found conjugated to hemoproteins such as hemoglobin (Hb), the most abundant pool of heme in mammals
endothelial barrier (EB) disruption is a hallmark of several inflammatory diseases [39,40,41,42], and studies in animal models suggest that this process is involved in the pathogenesis of acute complications of sickle cell disease (SCD), namely acute chest syndrome (ACS) [21, 22]
The most important result of our study was the demonstration that heme is capable to induce EB disruption even in the presence of serum proteins, but that this effect only occurs with serum from SCD patients, but not from healthy volunteers, and is associated with hemopexin levels

Summary

Introduction

Heme is a ubiquitous molecule present in almost all forms of life, that is normally found conjugated to hemoproteins such as hemoglobin (Hb), the most abundant pool of heme in mammals. FEH toxicity can be caused by direct (i.e., intercalation of heme in cellular membranes) or indirect (i.e., immune-mediated) mechanisms, and in regard to the latter, several studies demonstrated that heme can activate a myriad of innate immunity compartments such as TLR4-dependent pathways [5], neutrophil/neutrophil extracellular trap release [6, 7], complement [8, 9], inflammasomes [10], and hemostasis [11,12,13] Together, these studies support the notion that heme can act as a dangerassociated molecular pattern (DAMP) in diseases characterized by high hemolytic rates such as malaria, sepsis, hemolytic uremic syndrome and sickle cell disease (SCD), where FEH could trigger and/or contribute to the underlying inflammatory response [14,15,16,17].

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Conclusion