Date Published: February 2, 2017
Publisher: Public Library of Science
Author(s): Zoe Loomis, Paul Eigenberger, Katherine Redinius, Christina Lisk, Vijaya Karoor, Eva Nozik-Grayck, Scott K. Ferguson, Kathryn Hassell, Rachelle Nuss, Kurt Stenmark, Paul Buehler, David C. Irwin, Yunchao Su.
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.
Hemolysis induced release of hemoglobin (Hb) occurs in a host of patient populations including those suffering from hemolytic anemia, severe sepsis, or those prescribed chronic renal replacement therapy (CRRT) or extracorporeal membrane oxygenation (ECMO). Unless captured, sequestered, and compartmentalized by scavenging-proteins, Hb’s catalytic iron and globin chains become pathological mediators that contribute to morbidity associated with progression of lung and vascular diseases . For example, if not neutralized, Hb contributes to acute lung injury (ALI) from sepsis [2, 5], as well as, the more chronic vascular disease of pulmonary hypertension (PH) in sickle cell disease (SCD) [6–8]
The principle original finding of the present investigation was that Hb oxidation, cellular lipid peroxidation, and release of uncontained nucleic acids can trigger a TLR9 regulated IL-6 signaling pathway (Fig 6). In addition, these Hb-mediated events were inhibited when either Hb was bound to Hp or cells were co-treated with Hpx, suggesting oxidized Hb and released heme is an initiating event. Additionally, the data afforded from our co-culture model provides evidence that Hb-induced IL-6 upregulation in the endothelial cell contributes to increased metabolic activity in pulmonary smooth muscle cells, indicative of a pro-growth, pro-migration phenotype. Taken together these results suggest a novel pathway by which exposure to free Hb, due to chronic hemolysis, may contribute to pulmonary vascular disease.