Pericytes exist abundantly in the brain and compose the neurovascular unit. It has been elucidated that pericytes play a key role in the formation and maintenance of the blood-brain barrier (BBB), thus being considered to play significant roles in brain ischemia. The NADPH oxidase (Nox) family proteins are a major source of reactive oxygen species (ROS). We have reported previously that Nox4 is abundantly expressed in pericytes among the Nox family. Our goal was to elucidate the roles of Nox4 in brain pericytes during acute brain ischemia. We confirmed by quantitative PCR that Nox4 was abundantly expressed in human cultured brain microvascular pericytes (HBMPC) and was significantly upregulated by hypoxia. We produced a mouse middle cerebral artery occlusion (MCAO) stroke model and examined the expression of Nox4 in the brain. Immunofluorescent double labeling demonstrated that Nox4 expression was upregulated in microvessels particularly in peri-infarct areas and was co-stained with PDGFRβ, a pericyte marker. In order to elucidate the role of Nox4 in brain pericyte during brain ischemia, we generated mice with human Nox4 overexpression using a promoter of SM22α, a pericyte/smooth muscle cell marker (Tg-Nox4). We confirmed that SM22α was expressed in mouse brain pericytes by co-immunostaining with PDGFRβ. We isolated microvessels from Tg-Nox4 brain and confirmed that human Nox4 mRNA was highly expressed. In MCAO model, the infarct volume was significantly larger in Tg-Nox4 than in littermate controls. Confocal microscopy demonstrated that IgG leakage in peri-infarct areas was significantly increased in Tg-Nox4, suggesting that Nox4 overexpression in pericytes enhanced BBB breakdown during acute brain ischemia. To elucidate the mechanisms, we induced adenovirus-mediated overexpression of Nox4 in HBMPC. We demonstrated that Nox4 overexpression increased NFκB phosphorylation and MMP9 expression in the cells.In conclusion, Nox4 may be a major source of ROS in brain pericytes and is upregulated directly by hypoxia in peri-infarct areas during acute brain ischemia. Pericyte Nox4 may enhance BBB breakdown through the activation of NFκB-MMP9 signaling during acute brain ischemia.
Inflammation and the Emerging Role of the Toll-Like Receptor System in Acute Brain Ischemia
