Dysregulated Inflammation in Response to Hypoxia‐Confounded Injury

Abstract

Traumatic limb injury can cause extensive blood vessel damage that impairs oxygen and nutrient delivery to nearby tissues, resulting in a hypoxic state. The normal course of tissue repair involves a proper balance of pro‐ and anti‐inflammatory signaling; this balance is essential for tissue recovery as it aids in the destruction, containment or removal of pathogens and facilitates the repair of damaged tissue. However, in periods of extended hypoxia‐confounded injury, inflammation may become dysregulated. The mechanisms by which extended hypoxia may influence inflammatory cell activation during the acute recovery period are unknown. We therefore sought to create an in vitro wound model of sustained hypoxia to investigate inflammatory cell activation during this acute recovery period. We hypothesized that sustained hypoxia would result in greater immune cell activation over 72 hours compared to normoxia.We completed several experiments utilizing human monocytes (U937 cells), human dermal fibroblasts (HDFs), and human umbilical vascular endothelial cells (HUVECs) exposed to normoxia (~21% O2) or hypoxia (~1% O2) for 24, 48, or 72 hours. We sought to assess the impact of sustained hypoxia on cell proliferation (scratch wound assay) and immune cell activation (macrophage adhesion; transmigration of macrophages; expression of cell surface markers CD11b and CD14) at each of the time points listed above.We found that hypoxia impaired endothelial cell proliferation in a scratch wound assay, decreasing their ability to cover the gap area after 72 hours. We additionally found that macrophage transmigration was increased in response to hypoxia and that this result was correlated with increased immune cell proliferation as evidenced by increased cell surface expression of CD11b and CD14. We believe these results will provide valuable insight into the effect of sustained hypoxia on inflammatory signaling, and perhaps more importantly, the time course of signaling changes following hypoxia‐confounded injury.