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

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.

Total parasite biomass but not peripheral parasitaemia is associated with endothelial and haematological perturbations in Plasmodium vivax patients

“…Endothelial cell monolayer function was measured using ECIS, an electric cell-substrate impedance sensing system (ECIS Zθ, Applied BioPhysics, Troy, NY), as previously described 74 . The system then applies weak alternating currents through the electrode array and continuously measures the ability of the cell monolayer to impede the movement of electrons between adjacent endothelial cells (resistance).…”

Circulating Small Extracellular Vesicles May Contribute to Vaso-Occlusive Crises in Sickle Cell Disease

“…Several studies have implicated endothelial barrier disruption in the pathophysiology of sickle cell disease complications such as acute chest syndrome [37,38]. Evidence supports various molecules being released or generated by hemolysis (such as free hemoglobin, heme, and hemin) as possible mediators of this disruption [39][40][41][42]. While the plasma of our patients must contain these agents, we previously showed that the concentrations in our EV preparations were too low to affect our endothelial cells [32].…”

Endothelial superoxide dismutase 2 is decreased in sickle cell disease and regulates fibronectin processing

“…Sickle cell disease is characterized by episodes of increased hemolysis, which elevates levels of free hemoglobin and heme in the blood. 32 Challenging sickle mice with extracellular hemin, the oxidized form of heme, triggers acute chest syndrome, a pulmonary complication of SCD characterized by increased permeability of the lung vasculature. 33 We investigated whether simulating a hemolytic crisis by challenging siSOD2 hPMVECs with hemin would exacerbate differences in permeability.…”