Hemoglobin induced cell trauma indirectly influences endothelial TLR9 activity resulting in pulmonary vascular smooth muscle cell activation

Abstract

It is now well established that both inherited and acquired forms of hemolytic disease can promote pulmonary vascular disease consequent of free hemoglobin (Hb) induced NO scavenging, elevations in reactive oxygen species and lipid peroxidation. It has recently been reported that oxidative stress can activate NFkB through a toll-like receptor 9 (TLR9) mediated pathway; further, TLR9 can be activated by either nuclear or mitochondrial DNA liberated by stress induced cellular trauma. We hypothesis that Hb induced lipid peroxidation and subsequent endothelial cell trauma is linked to TLR9 activation, resulting in IL-6 mediated pulmonary smooth muscle cell proliferation. We examined the effects of Hb on rat pulmonary artery endothelial and smooth muscle cells (rPAEC and rPASMC, respectively), and then utilized TLR9 and IL6 inhibitors, as well as the Hb and heme binding proteins (haptoglobin (Hp) and hemopexin (Hpx), respectively) to further elucidate the aforementioned mediators. Further, we explored the effects of Hb in vivo utilizing endothelial cell (EC) specific myeloid differentiation primary response gene-88 (MyD88) and TLR9 null mice. Our data show that oxidized Hb induces lipid peroxidation, cellular toxicity (5.5 ± 1.7 fold; p≤0.04), increased TLR9 activation (60%; p = 0.01), and up regulated IL6 expression (1.75±0.3 fold; p = 0.04) in rPAEC. Rat PASMC exhibited a more proliferative state (13 ± 1%; p = 0.01) when co-cultured with Hb activated rPAEC. These effects were attenuated with the sequestration of Hb or heme by Hp and Hpx as well as with TLR9 an IL-6 inhibition. Moreover, in both EC-MyD88 and TLR9 null mice Hb-infusion resulted in less lung IL-6 expression compared to WT cohorts. These results demonstrate that Hb-induced lipid peroxidation can initiate a modest TLR9 mediated inflammatory response, subsequently generating an activated SMC phenotype.

Modulation of Functional Characteristics of Mesenchymal Stromal Cells by Acellular Preparation of Porcine Hemoglobin

“…In the internal milieu of erythrocytes, mammalian hemoglobin possesses a broad range of functions [24], which nowadays are well understood. However, when these biological macromolecules are present extracellularly, they exhibit many other still insufficiently elucidated biological functions [25][26][27]. This fact is increasing the added value of hemoglobin's use for biomedical and biotechnological purposes, especially in the case of xenogeneic ones.…”

Case series supporting heme detoxification via therapeutic plasma exchange in acute multiorgan failure syndrome resistant to red blood cell exchange in sickle cell disease

“…[1][2][3] Released free hemoglobin (Hb) and free heme activate neutrophils, peripheral blood mononuclear cells, and vascular endothelial cells via TLR-4, inducing pro-oxidant, inflammatory, coagulative, and cytotoxic damage. [4][5][6][7][8][9][10][11][12][13][14][15][16] Decreased nitric oxide bioavailability; extravascular translocation of heme; free radical generation with oxidative damage to lipids, proteins, and nucleic acids; and activation of inflammatory cascades 3,[17][18][19][20][21][22] contribute to SCD complications such as pulmonary and systemic vasculopathy, 23 pain crisis, and acute chest syndrome. 24 Free Hb is also associated with albuminuria, decreased glomerular filtration rate, and kidney damage in SCD patients.…”

“…In sickle cell disease (SCD), intravascular hemolysis accounts for one‐third of steady‐state hemolysis and increases even more during vaso‐occlusion . Released free hemoglobin (Hb) and free heme activate neutrophils, peripheral blood mononuclear cells, and vascular endothelial cells via TLR‐4, inducing pro‐oxidant, inflammatory, coagulative, and cytotoxic damage . Decreased nitric oxide bioavailability; extravascular translocation of heme; free radical generation with oxidative damage to lipids, proteins, and nucleic acids; and activation of inflammatory cascades contribute to SCD complications such as pulmonary and systemic vasculopathy, pain crisis, and acute chest syndrome .…”

Intrasurgical Protein Layer on Titanium Arthroplasty Explants: From the Big Twelve to the Implant Proteome

“…The exact cause‐and‐effect relationship for the extreme values of hemoglobin on the surface of implants has to be studied further in future experiments. Such studies are crucial, because of the known hemoglobin induced cell trauma on endothelial cells leading to endothelial cell permeability changes . Thus, hemoglobin on the implant surface may have negative effects on initial bone‐healing after implantation.…”