Affiliation:
1. Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, United States
2. Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States
3. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States
Abstract
Cell-free hemoglobin is a pathophysiological driver of endothelial injury during sepsis and acute respiratory distress syndrome (ARDS), but the precise mechanisms are not fully understood. We hypothesized that hemoglobin (Hb) increases leukocyte adhesion and endothelial activation in human lung microvascular endothelial cells (HLMVEC). We stimulated primary HLMVEC, or leukocytes isolated from healthy human donors, with Hb (0.5 mg/mL) and found that leukocyte adhesion to lung endothelium in response to Hb is an endothelial-dependent process. Next, we stimulated HLMVEC with Hb over time (1, 3, 6, and 24 h) and found increased transcription and release of inflammatory cytokines (IL-1β, IL-8, and IL-6). In addition, Hb exposure variably upregulated transcription, total protein expression, and cell-surface localization of adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Since VCAM-1 was most upregulated by Hb, we further tested mechanisms for Hb-mediated upregulation of VCAM-1 in HLMVEC. Although upregulation of VCAM-1 was not prevented by hemoglobin scavenger haptoglobin, heme scavenger hemopexin, or inhibition of nod-like receptor protein 3 (NLRP3) signaling, blocking Toll-like receptor 4 (TLR4) with small molecule inhibitor TAK-242 (1 µM) prevented upregulation of VCAM-1 in response to Hb. Consistently, Hb increased nuclear factor-κB (NF-κB) activation and intracellular reactive oxygen species (ROS), which were both prevented by TLR4 inhibition. Together, these data demonstrate that Hb increases leukocyte-endothelial adhesion and activates HLMVEC through TLR4 signaling, indicating a potential mechanism for Hb-mediated pulmonary vascular injury during inflammatory and hemolytic conditions.
Funder
Francis Family Foundation
HHS | NIH | National Heart, Lung, and Blood Institute
Publisher
American Physiological Society
Cited by
5 articles.
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