NAE1-Dependent Protein Neddylation Preserves Endothelial Identity and Vascular Integrity

Background

Endothelial dysfunction is a central driver of cardiovascular and inflammatory diseases, yet the post-translational mechanisms that preserve endothelial homeostasis remain incompletely understood. Protein neddylation, the covalent conjugation of a ubiquitin-like modifier, regulates diverse cellular processes, yet its physiological role in the vascular endothelium remains unknown. This study investigated whether protein neddylation is required to preserve endothelial identity and vascular homeostasis.

Methods

We generated tamoxifen-inducible endothelial-specific Nae1 knockout mice to inhibit neddylation and combined bulk RNA sequencing, single-cell and single-nucleus transcriptomics, quantitative proteomics, biochemical analyses, and gain- and loss-of-function approaches to define the role of endothelial neddylation in vascular homeostasis and inflammatory injury.

Results

Endothelial-specific Nae1 deletion caused rapid mortality associated with vascular leakage, platelet accumulation, inflammation, and multi-organ injury. Multi-omics analyses demonstrated profound loss of endothelial identity, characterized by suppression of core endothelial programs and activation of inflammatory, procoagulant, and pyroptotic pathways. Single-cell analyses revealed progressive endothelial dysfunction culminating in depletion of the endothelial population and remodeling of the vascular niche. Mechanistically, endothelial neddylation deficiency activated gasdermin D (GSDMD)- and gasdermin E (GSDME)-dependent pyroptosis, whereas dual inhibition of GSDMD and GSDME markedly attenuated inflammatory transcriptomic remodeling, vascular injury, hepatocyte death, immune cell infiltration, and platelet accumulation. Translational analyses demonstrated reduced endothelial neddylation in experimental endotoxemia and decreased expression of neddylation pathway components in human atherosclerosis and COVID-19 datasets. Conversely, restoration of endothelial neddylation partially reversed inflammatory endothelial transcriptomic reprogramming in vivo.

Conclusions

NAE1-dependent protein neddylation is an essential regulator of endothelial identity and vascular integrity. Loss of endothelial neddylation promotes gasdermin-dependent pyroptosis and thrombo-inflammatory vascular injury, whereas restoration of the neddylation pathway mitigates inflammatory endothelial dysfunction. These findings identify endothelial neddylation as a fundamental mechanism maintaining vascular homeostasis and a potential therapeutic target for cardiovascular and inflammatory diseases.

Clinical Perspective

What Is New?

• Endothelial-specific deletion of NAE1, the enzyme that initiates protein neddylation, causes rapid mortality in mice from multi-organ tissue damage, cell death, and inflammation.

• Neddylation loss activates the pyroptosis executioners GSDMD and GSDME, and silencing GSDMD and GSDME together reverses the transcriptomic and tissue-level damage caused by neddylation deficiency.

• The endothelial neddylation pathway is significantly downregulated in human clinical conditions, including atherosclerosis and COVID-19, as well as in experimental models of systemic inflammation.

What Are the Clinical Implications?

• Reduced neddylation in atherosclerotic human arteries suggests this pathway could serve as a biomarker or intervention point for endothelial dysfunction in cardiovascular disease.

• GSDMD and GSDME represent potential therapeutic targets for protecting endothelial integrity in vascular and inflammatory diseases, including endotoxemia.

• Because neddylation inhibitors such as MLN4924 (pevonedistat) are already in clinical use for cancer, these findings suggest a mechanism behind their reported vascular and hepatic toxicity that warrants monitoring.