NMDARs activation regulates endothelial ferroptosis via the PP2A-AMPK-HMGB1 axis

Vascular endothelial cells (VECs) are crucial for maintaining vascular health. VEC dysfunction contributes to various cardiovascular diseases. Ferroptosis, an iron-dependent form of regulated cell death, has been implicated in several pathologies, but its role in endothelial dysfunction remains unclear. N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ion channels involved in neuronal signaling. This study investigates the hypothesis that NMDAR activation induces ferroptosis in VECs, contributing to vascular damage. The researchers aimed to explore the underlying mechanisms and potential therapeutic targets to mitigate this process. Understanding this relationship holds promise for developing effective treatment strategies for vascular diseases.

The introduction mentions previous research highlighting the importance of VECs in maintaining vascular health and the role of VEC dysfunction in cardiovascular diseases. It also cites studies on ferroptosis, its mechanisms, and its involvement in various diseases. The role of NMDARs in neuronal signaling and their potential involvement in vascular processes is also mentioned. However, the paper doesn't provide a comprehensive review of existing literature on these topics, focusing instead on establishing the context and rationale for the current research.

The study employed both in vitro and in vivo approaches. In vitro experiments used human umbilical vein endothelial cells (HUVECs) treated with NMDA or GLU to activate NMDARs. The effects on ferroptosis were assessed through various methods, including RNA sequencing to identify involved pathways, transmission electron microscopy (TEM) to observe cellular morphology, MTT assays to measure cell viability, FerroOrange staining and iron content assays to determine iron levels, flow cytometry to measure lipid reactive oxygen species (LOS), and western blotting to analyze protein expression levels of ferroptosis biomarkers (GPX4, PTGS2) and components of the PP2A-AMPK-HMGB1 pathway. Ferroptosis inhibitors (Fer-1, Lip-1, DFO) were used to confirm the involvement of ferroptosis. The role of specific pathway components was further investigated using the PP2A inhibitor LB-100, AMPK activator AICAR, and HMGB1 siRNA. In vivo experiments used mice treated with NMDA or GLU, with or without inhibitors targeting the identified pathway, to assess the effects on ferroptosis in vascular tissue. Similar assays to the in vitro experiments were conducted on the mouse tissue. Statistical analyses were performed using GraphPad Prism 9.0.

In vitro studies showed that NMDAR activation significantly increased ferroptosis markers (iron content, MDA, LOS, decreased GSH, decreased GPX4, increased PTGS2) in HUVECs, an effect reversed by ferroptosis inhibitors. RNA sequencing implicated SLC7A11 and ferroptosis. The PP2A-AMPK-HMGB1 pathway was identified as a crucial regulator of NMDAR-induced ferroptosis; inhibition of PP2A, activation of AMPK, or knockdown of HMGB1 all mitigated ferroptosis. In vivo experiments demonstrated that NMDA or GLU induced ferroptosis, which was reversed by MK-801 (NMDAR inhibitor), LB-100, AICAR, and GLY (HMGB1 inhibitor), confirming the role of the PP2A-AMPK-HMGB1 pathway in NMDAR activation-induced ferroptosis in vivo. Erastin and RSL3 (ferroptosis inducers) were used to confirm that NMDAR is essential in the regulation of ferroptosis and that inhibition of NMDAR by MK-801 showed protective effects. The in vivo results mirror the in vitro findings, supporting the significant role of NMDAR in regulating endothelial ferroptosis via the PP2A-AMPK-HMGB1 pathway. Specific statistical values are presented in the figures and detailed in the methods section.

The findings demonstrate a novel mechanism by which NMDAR activation contributes to endothelial cell injury. The study suggests that NMDAR activation leads to the upregulation of PP2A activity and the downregulation of AMPK phosphorylation. This, in turn, increases HMGB1 expression, ultimately inducing ferroptosis. These findings are significant as they reveal a previously unknown mechanism linking NMDAR signaling to endothelial cell death via ferroptosis. Targeting this pathway could provide new avenues for therapeutic intervention in various vascular diseases. The study supports the potential therapeutic benefits of targeting NMDAR, PP2A, AMPK, or HMGB1 to prevent endothelial ferroptosis.

This study demonstrates for the first time that NMDAR activation induces endothelial ferroptosis through the PP2A-AMPK-HMGB1 pathway, both in vitro and in vivo. This discovery highlights the potential therapeutic benefits of targeting this pathway to prevent endothelial cell injury in various vascular diseases. Further research could focus on exploring the specific interactions within this pathway and investigating the clinical translation of these findings.

The study primarily focuses on HUVECs and C57BL/6 mice. The generalizability of these findings to other endothelial cell types and species requires further investigation. While the study provides strong evidence for the involvement of the PP2A-AMPK-HMGB1 pathway, it may not fully encompass all the intricate molecular mechanisms underlying NMDAR-induced ferroptosis. Further studies are needed to completely elucidate the precise interactions among these components.