Hemoglobin induced cell trauma indirectly influences endothelial TLR9 activity resulting in pulmonary vascular smooth muscle cell activation.
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.
Cardiovascular and Pulmonary Research Laboratory, Department of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, United States of America.
Zoe
Z
Loomis
Cardiovascular and Pulmonary Research Laboratory, Department of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, United States of America.
Paul
P
Eigenberger
Cardiovascular and Pulmonary Research Laboratory, Department of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, United States of America.
Katherine
K
Redinius
Cardiovascular and Pulmonary Research Laboratory, Department of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, United States of America.
Christina
C
Lisk
Cardiovascular and Pulmonary Research Laboratory, Department of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, United States of America.
Vijaya
V
Karoor
Cardiovascular and Pulmonary Research Laboratory, Department of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, United States of America.
Eva
E
Nozik-Grayck
Cardiovascular and Pulmonary Research Laboratory, Department of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, United States of America.
Scott K
SK
Ferguson
Division of Hematology and Colorado Sickle Cell Treatment and Research Center, University of Colorado-Denver School of Medicine, Aurora, Colorado, United States of America.
Kathryn
K
Hassell
Division of Hematology and Colorado Sickle Cell Treatment and Research Center, University of Colorado-Denver School of Medicine, Aurora, Colorado, United States of America.
Rachelle
R
Nuss
Cardiovascular and Pulmonary Research Laboratory, Department of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, United States of America.
Kurt
K
Stenmark
Division of Hematology, The Center for Biologics Evaluation and Research, United States Food and Drug Administration, Bethesda, Maryland, United States of America.
Paul
P
Buehler
Cardiovascular and Pulmonary Research Laboratory, Department of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, United States of America.
David C
DC
Irwin
D000743:Anemia, Hemolytic / Q000209:etiology; D000818:Animals; D049109:Cell Proliferation; D005260:Female; D006454:Hemoglobins / Q000378:metabolism*; D006976:Hypertension, Pulmonary / Q000209:etiology; D015850:Interleukin-6 / Q000502:physiology; D015227:Lipid Peroxidation; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular; D011651:Pulmonary Artery / Q000502:physiology*; D051217:Toll-Like Receptor 9 / Q000502:physiology*
D006454:Hemoglobins; D015850:Interleukin-6; C495370:Tlr9 protein, mouse; C489640:Tlr9 protein, rat; D051217:Toll-Like Receptor 9