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Aging-regulated PNUTS maintains endothelial barrier function via SEMA3B suppression

ABSTRACT: Age-related diseases pose great challenges to health care systems worldwide. During aging, endothelial senescence increases the risk for cardiovascular disease. Recently, it was described that Phosphatase 1 Nuclear Targeting Subunit (PNUTS) has a central role in cardiomyocyte aging and homeostasis. Here, we determined the role of PNUTS in endothelial cell aging. We confirmed that PNUTS is repressed in senescent endothelial cells (ECs). Moreover, PNUTS silencing elicits several of the hallmarks of endothelial aging: senescence, reduced angiogenesis and loss of barrier function. To validate our findings in vivo, we generated an endothelial-specific inducible PNUTS-deficient mouse line (Cdh5-CreERT2;PNUTSfl/fl), termed PNUTSEC-KO. Two weeks after PNUTS deletion, PNUTSEC-KO mice presented severe multiorgan failure and vascular leakage. Transcriptomic analysis of PNUTS-silenced HUVECs and lungs of PNUTSEC-KO mice revealed that the PNUTS-PP1 axis tightly regulates the expression of semaphorin 3B (SEMA3B). Indeed, silencing of SEMA3B completely restored barrier function after PNUTS loss-of-function. These results reveal a pivotal role for PNUTS in endothelial homeostasis through a SEMA3B downstream pathway that provides a potential target against the effects of aging in ECs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE263531 | GEO | 2024/04/14

REPOSITORIES: GEO

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Project description:ABSTRACT Rationale Premature senescence is conducive to aging and cardiovascular diseases. Nrf2 transcription factor, the master orchestrator of adoptive response to cellular stress, has been implicated in regulation of premature senescence in fibroblasts, neural and mesenchymal stem cells by transactivation of antioxidant gene expression. However, as we show here, human primary endothelial cells (ECs) devoid of Nrf2 and murine Nrf2 transcriptional knockout (tKO) aortas are senescent but do not encounter oxidative stress and damage, what contradicts this mechanism. Moreover, a molecular switch between normal, senescent and apoptotic fate remains unknown. Objective To elucidate the mechanism of Nrf2-related premature senescence of vascular system, to understand why Nrf2 deregulation does not cause oxidative stress exclusionary in ECs and to indicate a molecular switch determining ECs fate. Methods and Results Herein we evidence that ECs deficient in Nrf2 protein, or with limited Nrf2 activity in shear stress conditions, exhibit excessive S-nitrosylation of proteins. It is also a characteristic of Nrf2 tKO murine aortas, as determined by biotin switch assay in situ. Mass spectrometry analysis reveals that NOX4 is S-nitrosylated exclusively in ECs devoid of Nrf2. A functional role of S-nitrosylation is protection of ECs from death by inhibition of NOX4-mediated oxidative damage. As a result Nrf2-deficient ECs preserve oxidative balance but are redirected to premature senescence. The same phenotype is seen in Nrf2 tKO aortas. These effects are mediated by Keap1, a direct binding partner of Nrf2 and repressor of its transcriptional activity, remaining in cytoplasm unrestrained by Nrf2. S-nitrosylation, followed by senescence, can also be triggered in smooth muscle cells (SMCs) by EC-derived paracrine induction of iNOS. Conclusions Collectively, Keap1-dependent S-nitrosylation of NOX4 hampers oxidative detriment in ECs with disturbed Nrf2 signaling and may provide defence in the adjacent aortic cells. Overabundance of unrestrained Keap1 in the cytoplasm determines fate of ECs.

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Aging-regulated PNUTS maintains endothelial barrier function via SEMA3B suppression

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