Hypoxia Regulates Endothelin‐1 Production and Microglial Cell Activation

Abstract

Oxidative stress and inflammatory responses play critical roles in hypoxic‐ischemic brain injury.Microglial cells are rapidly activated in response to injury and stressful stimuli, including hypoxia.Endothelin‐1 (ET‐1) is a potent vasoconstrictor that has been associated with cerebrovascular diseases. Hypoxia stimulates endothelial ET‐1 production. However, the role of ET‐1 in microglia under hypoxia is not clear. The aim of this project was to characterize the effect of hypoxia in a human microglial cell line, HMC3. We induced hypoxia using a chamber (1% O2, 5% CO2, and 92 % N2) at 37 °C for 4h. MUSE Oxidative Stress Assay was performed to measure reactive oxygen species (ROS) formation, ELISA to determine TNF, IL‐6, and ET‐1 levels, qPCR to measure gene expression of ET‐1, and immunofluorescence staining to visualize and compare the presence of ET‐1 in hypoxic and controlgroup cells. It was observed that, when compared to normoxic HMC3 cells, hypoxic HMC3 exposure significantly increased the ROS by a factor of 2.5 (p<0.001, n=3), the pro‐inflammatory cytokine TNF‐α increased 3.8 times (p<0.01, n=4), and IL‐6 increased by a factor of 1.6 (p<0.01, n=4). In addition, hypoxia stimulates ET‐1 gene expression 5.0‐fold (p<0.001, n=4) and increased protein production 1.3 times (p<0.01, n=4). Consequently, treatment with ET‐1 increased the amount of ROS, TNF‐α, and IL‐6 in HMC3 cells by a factor of 1.4 (p<0.05, n=4), 1.6 (p<0.001, n=4), and 1.9 (p<0.05, n=4), respectively. All these events were blocked by ET‐1 receptor A (ETRA) antagonist, BQ123. Our results suggest that hypoxic conditions create a cycle of microglial cell activation leading to increased ROS and ET‐1 production that further stimulate microglial cells. Thus, we posit that the ET‐1 receptor blockade represents a promising therapeutic approach to regulate microglial cell responses in hypoxic‐ischemic brain injury.