The Mechanism of Vascular Endothelial Dysfunction Induced by Ferroptosis Mediated by NARFL Knockout
Authors: Hu Hui , Luo Jing , Yu Li , Qi Daoxi , Li Boyu , Cheng Yating , Wang Chen , Zhang Xiaokang , Lou Qiyong , Zhai Gang , Ruan Yonglin , Huang Jianfei , Shi Shengchi , Yin Zhan , Zheng Fang 
Published: February 2024

BACKGROUND

Nuclear prelamin A recognition factor-like (NARFL) plays a crucial role in cytosolic iron-sulfur protein assembly (CIA) and protects cells against oxidative stress. In our previous study, we identified a novel homozygous mutation in NARFL that led to decreased expression in a consanguineous family with diffuse pulmonary arteriovenous malformations (DPAVMs) secondary to pulmonary hypertension. Additionally, we observed that narfl deletion in zebrafish resulted in larvae lethality, subintestinal vessel malformation, and increased oxidative stress. In this study, we aimed to further investigate the function of NARFL and elucidate the pathological manifestations of NARFL deficiency in zebrafish models, cellular models, mouse models, and clinical samples, focusing on the underlying molecular mechanisms.

METHODS

We observed the behavioral and phenotypic abnormalities in zebrafish caused by narfl deletion and investigated the mechanism behind vascular morphological abnormalities. Furthermore, we constructedNARFLgene knockout stable cell lines in human pulmonary microvascular endothelial cells (HPMEC) to examine the morphological and functional changes in endothelial cells caused by NARFL deletion. We studied the effects of NARFL deletion on ferroptosis and its potential rescue using a ferroptosis inhibitor. To investigate the function of the human NARFL homolog Ciao3 gene in vascular development, we created a mouse model with a knockout of theCiao3gene. Finally, we compared the distribution of tagSNPs of NARFL using the SNaPshot method between cases and controls to confirm the role of the Ciao3 gene in endothelial dysfunction.

RESULTS

Narfl deletion in zebrafish resulted in larvae lethality, vascular malformation with abnormal blood flow, abnormal blood-brain barrier (BBB) structure, and brain neuron lesions. Fluorescence probe detection showed increased iron, enhanced oxidative stress, lipid peroxidation, and decreased mitochondrial respiration in response to narfl deficiency, which could be partially alleviated by the use of the ferroptosis inhibitor Ferrostatin-1. We observed downregulation of the iron-sulfur protein cyp2p8 expression in blood vessels of narfl-deficient zebrafish through qRT-PCR and WISH experiments. In HPMEC cells, NARFL deficiency resulted in decreased proliferation, abnormal mitochondrial morphology, increased levels of iron and oxidative stress, and decreased mitochondrial respiration. Functional experiments on endothelial cells revealed decreased tube formation ability and enhanced permeability in response to NARFL deficiency. WB experiments showed downregulation of GPX4, SLC7A11, and Ferritin, while TFR1 and IRP1 were upregulated. Downregulation of NARFL also affected the expression of the iron-sulfur protein CYP2J2. Co-IP results indicated that NARFL deletion led to incompatibility among the CIA system-associated proteins. In mice, Ciao3 deletion in the embryonic stage resulted in embryonic death, vascular dysplasia, impaired differentiation of endothelial progenitor cells, and abnormalities in the expression of ferroptosis-related proteins. Reduction of Ciao3 impaired vascular function and decreased ring formation ability in adult heterozygous mice.NARFLpolymorphisms rs11248948, rs2071952, and rs611289 were identified as susceptible sites for epilepsy, while rs11792680 was associated with susceptibility to pulmonary hypertension, epilepsy, and neurodegenerative diseases.

CONCLUSION

NARFL knockout disrupts its interaction with CIA system-related proteins, leading to decreased aconitase activity, increased IRP1 activity, endothelial cell ferroptosis pathway abnormalities, enhanced ferroptosis and oxidative stress, and ultimately vascular endothelial dysfunction. This dysfunction is responsible for the death of embryos innarfl-/-zebrafish andCiao3-/-mice, as well as the susceptibility to pulmonary hypertension, epilepsy, and neurodegenerative diseases.

What Is New?

Elucidation of the mechanism behind NARFL knockout-induced death through dynamic visualization experimentsin vivoand mechanism and function experimentsin vitro:The study explored the function of NARFL, as it is known as a “knockout lethal” protein. Bothin vivoandin vitroexperiments have confirmed that NARFL acts as the “transmitter” of cytoplasmic iron-sulfur clusters. Its absence prevents interaction with associated proteins of the CIA system, leading to reduced cisaconitase activity, enhanced IRP1 activity, ferroptosis of endothelial cells, and increased oxidative stress, eventually resulting in cell death.

Providing new research ideas for the study of cytoplasmic iron-sulfur proteins: Most current studies focus on the function of mitochondrial iron-sulfur proteins and their relationship with iron death. However, research on extramitochondrial iron-sulfur proteins is relatively limited. This study provides data support and research ideas for understanding the function of extramitochondrial iron-sulfur proteins by exploring the pathological mechanism of NARFL and the mediation of iron-sulfur protein maturation.

What Are the Clinical Implications?

From rare diseases to common diseases: Through the investigation of the lethal mechanism of NARFL knockout and the study ofNARFLgene polymorphisms associated with vascular endothelial dysfunction diseases, we propose the hypothesis that NARFL may be a susceptibility gene for these diseases. This study provides data support for the hypothesis and contributes to our understanding of the role of NARFL in vascular endothelial dysfunction diseases.