PEN2 stabilizes SLC25A51 to inhibit vascular calcification through NAD+ redistribution between mitochondrion and nucleus

Background Arterial calcification is an active, cell-regulated process and a major contributor to cardiovascular morbidity, particularly in ageing and chronic kidney disease. Although nicotinamide adenine dinucleotide (NAD⁺) has emerged as a key metabolic regulator in cardiovascular biology, clinical strategies aimed at globally increasing NAD⁺ levels have yielded inconsistent results, suggesting that subcellular NAD⁺ distribution rather than total abundance may be critical. The mechanisms governing NAD⁺ compartmentalization during vascular calcification remain undefined. Purpose Presenilin enhancer 2 (PEN2), a core component of the γ-secretase complex and recently identified molecular target of metformin, has not been investigated in vascular aging. We therefore aimed to determine whether PEN2 functions as an endogenous regulator of vascular calcification by modulating mitochondrial–nuclear NAD⁺ redistribution through stabilization of the mitochondrial NAD⁺ transporter SLC25A51. Methods Global PEN2 haploinsufficient mice, VSMC-specific PEN2 knockout mice, and PEN2-overexpressing models were generated to define the role of PEN2 in vascular calcification in vivo. Human VSMCs were exposed to pro-calcifying conditions with loss- and gain-of-function approaches applied in vitro. Calcification was assessed by Alizarin Red staining, calcium quantification, and osteogenic marker analysis. Subcellular NAD⁺ levels in mitochondrial and nuclear fractions were quantified using enzymatic cycling assays. Transcriptomic profiling, protein–protein interaction analysis, co-immunoprecipitation, ubiquitination assays, and mass spectrometry were performed to identify PEN2-interacting partners and delineate downstream mechanisms. Results PEN2 expression was markedly reduced in radial artery tissues from patients with VC. Both global PEN2+/− and VSMCs–specific deletion of PEN2 significantly exacerbated vascular calcification, whereas PEN2 overexpression markedly attenuated VC progression. Integrative analyses combining transcriptomic profiling and protein–protein interactome mapping identified SLC25A51, a newly characterized mitochondrial NAD+ transporter, as a direct interacting partner of PEN2. Mechanistically, PEN2 stabilizes SLC25A51 to maintain mitochondrial NAD⁺ pools and prevent aberrant nuclear NAD⁺ accumulation, thereby preserving mitochondrial respiration and restraining pathological nuclear signaling. Loss of this regulatory axis leads to PARP1 overactivation and subsequent induction of osteogenic transcriptional programs, including RUNX2 and BMP2, ultimately promoting VSMCs osteogenic transdifferentiation and VC. Conclusion These findings uncover a previously unrecognized PEN2–SLC25A51–NAD⁺ signaling axis that governs subcellular NAD⁺ compartmentalization and mitochondrial integrity during VC. Targeting this pathway to restore NAD⁺ homeostasis may represent a promising therapeutic strategy for preventing or treating vascular calcification.abstract scheme