Hemoglobin-Induced Endothelial Cell Permeability Is Controlled, in Part, via a Myeloid Differentiation Primary Response Gene–88–Dependent Signaling Mechanism

Abstract

The release of hemoglobin (Hb) with hemolysis causes vascular dysfunction. New evidence implicates Hb-induced NF-kB and hypoxia inducible factor (HIF) activation, which may be under the control of a Toll-like receptor (TLR)-signaling pathway. Nearly all TLR-signaling pathways activate the myeloid differentiation primary response gene-88 (MyD88) that regulates NF-kB. We hypothesized that the differing transition states of Hb influence endothelial cell permeability via NF-kB activation and HIF regulation through a MyD88-dependent pathway. In cultured human dermal microvascular endothelial cells (HMECs-1), we examined the effects of Hb in the ferrous (HbFe 21 ), ferric (HbFe 31 ), and ferryl (HbFe 41 ) transition states on NF-kB and HIF activity, HIF-1a and HIF-2a mRNA upregulation, and monolayer permeability, in the presence or absence of TLR4, MyD88, NF-kB, or HIF inhibition, as well as superoxide dismutase (SOD) and catalase. Our data showed that cell-free Hb, in each transition state, induced NF-kB and HIF activity, up-regulated HIF-1a and HIF-2a mRNA, and increased HMEC-1 permeability. The blockade of either MyD88 or NF-kB, but not TLR4, attenuated Hb-induced HIF activity, the up-regulation HIF-1 and HIF-2a mRNA, and HMEC-1 permeability. The inhibition of HIF activity exerted less of an effect on Hb-induced monolayer permeability. Moreover, SOD and catalase attenuated NF-kB, HIF activity, and monolayer permeability. Our results demonstrate that Hb-induced NF-kB and HIF are regulated by two mechanisms, either MyD88 activation or Hb transition state-induced ROS formation, that influence HMEC-1 permeability.Keywords: hemolytic disease; NF-kB; Toll-like receptors; MyD88; sicklecell disease Humans with chronic vascular inflammation and vasoconstriction develop progressive endothelial dysfunction and vascular diseases (e.g., pulmonary arterial hypertension, atherosclerosis, and sickle-cell disease) (1, 2). Vascular diseases develop in the face of chronic hemolysis, as occurs with hemoglobinopathies (including sickle-cell disease), or with the intermittent release of low to moderate levels of plasma-free hemoglobin (Hb), as may be seen in atherosclerosis with intraplaque hemorrhage.Low to moderate levels of plasma-free Hb may, in part, mediate these diseases (1-4). However, studies to date have not fully addressed the complete mechanisms responsible for Hb-induced vascular dysfunction.The a 2 b 2 tetramers of Hb transition safely and effectively between the relaxed (oxy) and tense (deoxy) conformational states, and the oxidation of Hb is controlled by red blood cell catalase, superoxide dismutase (SOD), and ferric Hb reductase. However, after their release from the red blood cell, Hb tetramers and dimers can be oxidized from ferrous (HbFe 21 ) to ferric (HbFe 31 ) (3-5). In theory, HbFe 31 in local tissue environments may react with hydrogen peroxide (H 2 O 2 ) to transition between the ferryl (HbFe 41 ) and HbFe 31 states, leading to heme release, globin chain denaturation, and globin chain crosslinking (4, 5). Oxid...

Cell-free hemoglobin promotes primary graft dysfunction through oxidative lung endothelial injury

“…The current translational studies suggest that CFH contributes to increased microvascular permeability in PGD. While other studies have reported associations between circulating CFH and alterations in endothelial function in models of atherosclerosis, malaria, and sickle cell disease (38)(39)(40), there have been few prior studies of the direct effects of CFH on microvascular endothelial permeability (41). Oxidized CFH, which is present in atherosclerotic plaques, results in intercellular gap formation and increased permeability in cultured endothelial cell monolayers (42,43).…”

“…Oxidized CFH, which is present in atherosclerotic plaques, results in intercellular gap formation and increased permeability in cultured endothelial cell monolayers (42,43). Hypoxia-responsive genes including HIF have also been implicated in hemoprotein-induced endothelial permeability in dermal microvascular endothelial cells (41).…”

Oxidized Ferric and Ferryl Forms of Hemoglobin Trigger Mitochondrial Dysfunction and Injury in Alveolar Type I Cells

“…However, incubation with HbFe 31 showed no hyperpolarization over untreated control ( Figure 5A). Exposure to HbFe ORIGINAL RESEARCH (38,39). A recent study ruled out any significant contribution of metHb-iron and redox-mediated reactions in human lung AT2 cells (31).…”

Dynamic interactions ofPlasmodiumspp. with vascular endothelium

“…104 In addition, the barrier disruptive effects of heme could be mediated by NLRP3, 105 TLR4 106 or other MyD88 signaling pathways. 107 Heme induced inflammatory effects can be inhibited by hemoglobin-and heme-binding proteins such as haptoglobin 108 and hemopexin, 109 or detoxification of hemoglobin breakdown products by nitric oxide, 110 heme oxygenase-1 or carbon monoxide which converts toxic methemoglobin to carboxyhemoglobin. 111 …”