Increased NOX2 expression in astrocytes leads to eNOS uncoupling through dihydrofolate reductase in endothelial cells after subarachnoid hemorrhage

by Selleckchem | Sep 13 2023

Introduction: Endothelial nitric oxide synthase (eNOS) uncoupling plays a significant role in acute vasoconstriction during early brain injury (EBI) after subarachnoid hemorrhage (SAH). Astrocytes in the neurovascular unit extend their foot processes around endothelia. In our study, we tested the hypothesis that increased nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) expression in astrocytes after SAH leads to eNOS uncoupling.

Methods: We utilized laser speckle contrast imaging for monitoring cortical blood flow changes in mice, nitric oxide (NO) kits to measure the level of NO, and a co-culture system to study the effect of astrocytes on endothelial cells. Moreover, the protein levels were assessed by Western blot and immunofluorescence staining. We used CCK-8 to measure the viability of astrocytes and endothelial cells, and we used the H2O2 kit to measure the H2O2 released from astrocytes. We used GSK2795039 as an inhibitor of NOX2, whereas lentivirus and adeno-associated virus were used for dihydrofolate reductase (DHFR) knockdown in vivo and in vitro.

Results: The expression of NOX2 and the release of H2O2 in astrocytes are increased, which was accompanied by a decrease in endothelial DHFR 12 h after SAH. Moreover, the eNOS monomer/dimer ratio increased, leading to a decrease in NO and acute cerebral ischemia. All of the above were significantly alleviated after the administration of GSK2795039. However, after knocking down DHFR both in vivo and in vitro, the protective effect of GSK2795039 was greatly reversed.

Discussion: The increased level of NOX2 in astrocytes contributes to decreased DHFR in endothelial cells, thus aggravating eNOS uncoupling, which is an essential mechanism underlying acute vasoconstriction after SAH.

Comments:

The findings from this study suggest that increased expression of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) in astrocytes following subarachnoid hemorrhage (SAH) leads to endothelial nitric oxide synthase (eNOS) uncoupling. eNOS uncoupling is known to play a significant role in acute vasoconstriction during early brain injury (EBI) after SAH.

To investigate this hypothesis, the researchers employed several methods. They used laser speckle contrast imaging to monitor changes in cortical blood flow in mice. Nitric oxide (NO) kits were used to measure the level of NO, and a co-culture system was utilized to study the effect of astrocytes on endothelial cells. Protein levels were assessed using Western blot and immunofluorescence staining. The viability of astrocytes and endothelial cells was measured using CCK-8, while the H2O2 released from astrocytes was measured using an H2O2 kit. The researchers also employed GSK2795039, an inhibitor of NOX2, and performed dihydrofolate reductase (DHFR) knockdown using lentivirus and adeno-associated virus both in vivo and in vitro.

The results of the study showed that the expression of NOX2 and the release of H2O2 in astrocytes were increased after SAH, accompanied by a decrease in endothelial DHFR 12 hours after the event. This led to an increase in the eNOS monomer/dimer ratio, resulting in a decrease in NO production and subsequent acute cerebral ischemia. Administration of GSK2795039 significantly alleviated these effects. However, when DHFR was knocked down, the protective effect of GSK2795039 was greatly reversed.

The authors discuss that the elevated NOX2 levels in astrocytes contribute to decreased DHFR in endothelial cells, thereby exacerbating eNOS uncoupling. This mechanism is crucial in understanding the acute vasoconstriction observed after SAH. The study sheds light on the role of astrocytes in the neurovascular unit and their impact on endothelial function, providing insights into potential therapeutic targets for managing SAH-induced cerebral ischemia.