biostudies-arrayexpressBowei LiMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-17006Age-related microvascular dysfunction disrupts nutrient homeostasis and waste clearance, leading to organ failure. However, a critical gap remains in our understanding of the specific molecular drivers of vascular deterioration and how they orchestrate organism-wide aging. Here, we identify progressive activation of the CCAAT/enhancer-binding protein β (C/EBPβ)/asparagine endopeptidase (AEP) pathway in aging vascular endothelial cells contributes to vascular degeneration and lifespan reduction. Endothelial-specific C/EBPβ or AEP overexpression accelerated vascular aging and shortened lifespan in mice. Mechanistically, AEP mediates proteolytic cleavage of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (oxidised form, NAD+) biosynthesis, resulting in systemic NAD⁺ depletion and senescence that elicits both central and peripheral vascular dysfunction and ultimately systemic aging. Genetic ablation of AEP or expression of AEP-resistant NAMPT N136A mutant significantly ameliorated vascular aging and extended lifespan in endothelial-specific Tie 2-C/EBPβ transgenic mice. Pharmacologically, AEP inhibitor CP#11A or nicotinamide mononucleotide (NMN) supplementation alleviated age-related vascular decline, with CP#11A showing superior efficacy. These findings support a model in which endothelial senescence contributes to vascular dysfunction and systemic aging mediated by C/EBPβ/AEP signaling, and establish C/EBPβ/AEP as a therapeutic target to preserve vascular integrity and mitigate systemic frailty in aging populations.biostudies-arrayexpressLibrary Construction - Sequencing libraries were constructed using the Chromium Single Cell 3’ v3.1 reagent kit (10X Genomics).Sample Collection - Mice were euthanized, and quadriceps femoris, brain, and liver were dissected. Magnetic‑activated cell sorting (MACS) of CD31+ endothelial cells: Cells were centrifuged at 400g for 5 min, and the pellet was resuspended in 80 µL of buffer (PBS with 0.5% BSA and 2 mM EDTA), followed by addition of 20 µL of CD45 microbeads. After a 15‑minute incubation at 4°C in the dark, cells were centrifuged at 300g for 10 min. The cell suspension was loaded onto an LS column pre‑rinsed with 1 mL of buffer. The CD45‑negative flow‑through was collected, and the column was washed with 2 mL of buffer. The CD45‑negative fraction was centrifuged at 400g for 5 min, resuspended in 90 µL of buffer with 10 µL of CD31 microbeads, and incubated for 15 min at 4°C in the dark. After centrifugation at 300g for 10 min, cells were resuspended in 1 mL of buffer and applied to a fresh LS column. The column was washed with 2 mL of buffer, then removed from the magnet. CD31‑positive endothelial cells were eluted by flushing with 4 mL of buffer using the plunger. The eluted cells were centrifuged at 400g for 5 min and resuspended in RPMI‑1640 for downstream applications.Sequencing - The sequencing was carried out on the Illumina NovaSeq X platform according to the manufacturer's instructions.Nucleic Acid Extraction - After MACS sorting, purified CD31+ endothelial cells were collected by centrifugation. Cell pellets were lysed using the proprietary lysis buffer provided in the Chromium Single Cell 3' v3.1 kit (10X Genomics). RNA was released from the lysed cells and subsequently reverse-transcribed into cDNA within the Gel Bead-in-Emulsions (GEMs). The resulting barcoded cDNA was then purified and amplified for downstream library construction.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Raw gene expression counts were normalized and transformed using the Seurat package (version 4.0) in R. The \""LogNormalize\"" method was applied using the NormalizeData function, where each cell's gene expression measurements were divided by the total number of transcripts in that cell, multiplied by a scale factor of 10,000, and then natural-log transformed. Following normalization, the 2000 most highly variable genes were identified using the FindVariableFeatures function with the variance stabilizing transformation (\""vst\"") method. The normalized and scaled data matrix was used for downstream dimensionality reduction (PCA), clustering (Louvain algorithm), and visualization (UMAP). Differential expression analysis between sample groups was performed using the FindMarkers function with the Wilcoxon rank-sum test, and gene expression values were log-normalized for visualization purposes.UnknownTranscriptomicsGenomicsProteomics10X Chromium ControllerIllumina NovaSeq XRNA-seq of coding RNA from single cellsMus musculusEndothelial C/EBPβ/AEP Pathway Drives Vascular Deterioration and Systemic AgingBowei Li, Shuke Nie, Mengmeng Wang, Jiawei An, Zhengjiang Qian, Xin Meng, Laura E. Edgington-Mitchell, Zhentao Zhang, Xifei Yang and Keqiang YeBowei LifalseEndothelial C/EBPβ/AEP Pathway Drives Vascular Deterioration and Systemic AgingAge-related microvascular dysfunction disrupts nutrient homeostasis and waste clearance, leading to organ failure. However, a critical gap remains in our understanding of the specific molecular drivers of vascular deterioration and how they orchestrate organism-wide aging. Here, we identify progressive activation of the CCAAT/enhancer-binding protein β (C/EBPβ)/asparagine endopeptidase (AEP) pathway in aging vascular endothelial cells contributes to vascular degeneration and lifespan reduction. Endothelial-specific C/EBPβ or AEP overexpression accelerated vascular aging and shortened lifespan in mice. Mechanistically, AEP mediates proteolytic cleavage of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (oxidised form, NAD+) biosynthesis, resulting in systemic NAD⁺ depletion and senescence that elicits both central and peripheral vascular dysfunction and ultimately systemic aging. Genetic ablation of AEP or expression of AEP-resistant NAMPT N136A mutant significantly ameliorated vascular aging and extended lifespan in endothelial-specific Tie 2-C/EBPβ transgenic mice. Pharmacologically, AEP inhibitor CP#11A or nicotinamide mononucleotide (NMN) supplementation alleviated age-related vascular decline, with CP#11A showing superior efficacy. These findings support a model in which endothelial senescence contributes to vascular dysfunction and systemic aging mediated by C/EBPβ/AEP signaling, and establish C/EBPβ/AEP as a therapeutic target to preserve vascular integrity and mitigate systemic frailty in aging populations.2026-05-30T00:00:00Z2026-05-30T01:01:26.832Z2026-05-06T12:18:19.702ZE-MTAB-17006ERP193076EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184