Abstract
Thoracic and abdominal aortic aneurysm poses a substantial mortality risk in adults, yet many of its underlying factors remain unidentified. Here, we identify mitochondrial nicotinamide adenine dinucleotide (NAD)⁺ deficiency as a causal factor for the development of aortic aneurysm. Multiomics analysis of 150 surgical aortic specimens indicated impaired NAD+ salvage and mitochondrial transport in human thoracic aortic aneurysm, with expression of the NAD+ transporter SLC25A51 inversely correlating with disease severity and postoperative progression. Genome-wide gene-based association analysis further linked low SLC25A51 expression to risk of aortic aneurysm and dissection. In mouse models, smooth muscle-specific knockout of Nampt, Nmnat1, Nmnat3, Slc25a51, Nadk2 and Aldh18a1, genes involved in NAD+ salvage and transport, induced aortic aneurysm, with Slc25a51 deletion producing the most severe effects. Using these models, we suggest a mechanism that may explain the disease pathogenesis: the production of type III procollagen during aortic medial matrix turnover imposes a high demand for proline, an essential amino acid component of collagen. Deficiency in the mitochondrial NAD⁺ pool, regulated by NAD⁺ salvage and transport, hinders proline biosynthesis in mitochondria, contributing to thoracic and abdominal aortic aneurysm.
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Data availability
The bruker.raw files of the proteome datasets can be obtained from the PRIDE database (https://www.ebi.ac.uk/pride/archive, accession number PXD043052). RNA-seq gene expression profiles can be obtained from the Gene Expression Omnibus (accession number GSE235161). The raw data files for human and mouse aortic metabolites were deposited in MetaboLights (https://www.ebi.ac.uk/metabolights/index, accession number MTBLS8062). Source data are provided with this paper.
Code availability
No custom codes were used during this study. All packages used are described in the Methods.
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Acknowledgements
We thank staff at the Department of Laboratory Animal Science of Shanghai Medical College, Fudan University for their assistance with animal experiments. We thank staff at the Core Facility of Shanghai Medical College, Fudan University for their assistance with proteomics and C. Shen at Shanghai Omicsolution for his assistance with metabolomics. This work was supported by the National Natural Science Foundation of China (51927805 (K.Z.), 82370472 (W.Z.) and 82070482 (W.Z.)), the Shanghai Municipal Science and Technology Major Project 2017SHZDZX01 (W.Z.), the Science and Technology Commission of Shanghai Municipality (17JC1400200 (W.Z.) and 23YF1405800 (W.M.)). We acknowledge the use of ChatGPT, an AI language model developed by OpenAI, for its assistance in polishing the grammar, spelling and sentence structure of the manuscript.
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W.Z. directed and designed research; K.Z., W.M., M.A., H.L. and C.W. coordinated acquisition and quality evaluation of human aortic tissue samples; J. Zhang and W.M. analyzed clinical characteristics of patients; J. Zhang and G.Y. performed MS-based proteomic experiments; J. Zhang and F.S. performed MRM-based metabolomic experiments; J. Zhang, D.Y., F.Y. and W.Z. performed analyses of proteomic, transcriptomic and metabolomic data; S. Zheng and C.P. performed genome-wide association meta-analysis; J. Zhang, Y.T., S. Zhang, Z.X., S.L., Y.F. and W.M. performed histopathological assessments; Y.T. performed immunohistochemistry experiments; J. Zhang, Y.T., S. Zhang, Z.X., S.L., C.H., Y.L., Y. Xin, J. Zhu, W.H. and W.W. performed mouse experiments; W.Z., J. Zhang, Y. Xu and W.M. wrote and revised the manuscript; all authors discussed and commented on the manuscript.
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Extended data
Extended Data Fig. 1 Comparative proteomics analysis.
a, Distribution of log2-transformed abundance of identified proteins passing quality control. b, Cumulative number of protein identifications. c, Box plots represent the distribution of the identified protein counts across patient subgroups: degenerated (n = 110 patients) and healthy (n = 34 patients), male (n = 105 patients) and female (n = 39 patients), with (n = 56 patients) or without (n = 88 patients) hypertension, and with higher (n=44) or lower (n=47) aortic architecture scores. d, Principal component analysis of the proteomics data. Please see Methods for aortopathy risk proteins. e, Volcano plots of differentially expressed proteins in groups categorized by aortic degeneration and aortic architecture scores. f, Scatter plots showing differentially expressed proteins across the same comparisons, highlighting downregulation of SLC25A51. g, Functional enrichment analyses based on aortic degeneration and aortic architecture scores. Bar and line plots denote counts of genes involved in pathways and -log10-transformed p-values, respectively. Pathway activation or inhibition is represented by z-scores. Box plots are represented as median and interquartile range, whiskers extend to the minimum and maximum values, p-values were determined by Mann-Whitney U test or two-tailed Studentʼs t-tests, and Benjamini-Hochberg method was used to correct for multiple comparisons.
Extended Data Fig. 2 Comparative transcriptomic analysis and comparative targeted metabolomic analysis.
a, Overview of the global and mitochondrial genes characterization in aortic specimens, stratified by the degenerative status, age, sex, and hypertension. The dashed curves fitted by loess regression, show the distribution of protein identifications among the specimens. The shaded area beneath the curves represents 95% confidence intervals. b, Cumulative number of gene identifications. c, Box plots represent the distribution of the identified protein counts across patient subgroups: degenerated (n = 31 patients) and healthy (n = 11 patients), male (n = 35 patients) and female (n = 7 patients), with (n = 14 patients) or without (n = 28 patients) hypertension, and with higher (n = 10 patients) or lower (n = 14 patients) aortic architecture scores. d, Principal component analysis of the transcriptomics data. Please see Methods for aortopathy risk genes. e, Volcano plots of differentially expressed genes in degenerated vs. healthy aorta groups. f, Pathway alternations in degenerated vs. healthy aorta groups. Bar and line plots denote counts of genes involved in the pathway and -log10-transformed p-values, respectively. The z-scores represent the predicted state of activation or inhibition in the identified pathways. Up- and down-regulated pathways are indicated in red and blue, respectively. g, Principal component analysis of 261 targeted metabolites. h, Scatterplot that shows statistical significance (VIP scores) vs. magnitude of change (fold change) of differential metabolites in degenerated vs. healthy aorta groups. Box plots are represented as median and interquartile range, whiskers extend to the minimum and maximum values, p-values were determined by limma statistical analysis, Benjamini-Hochberg method was used to correct for multiple comparisons. VIP, variable importance in projection.
Extended Data Fig. 3 Immunoblot validation, correlation analysis, and comparison of protein and NAD+ levels in aortic tissues categorized by degeneration and architecture scores.
a, Western blots of NAD+ metabolism-related proteins and differentiating markers of smooth muscle cells. b, Western blot using anti-acetylated lysine protein antibody in healthy (n = 6 patients) and degenerated (n = 7 patients) groups. SIRT3, NAMPT, NMNAT1, and SLC25A51 are proteins involved in NAD+ consumption, salvage, and mitochondrial transportation. TAGLN and MMP2 are markers of contractile status and synthetic status, respectively. Patient age and relative intensity of blots are marked below the blot stripes. Correlation between protein abundances of SLC25A51 and NAMPT (c), and SLC25A51 and NMNAT1 (d). NMNAT3 protein was not detected by LC-MS based characterization. Correlation between aortic architecture score and COL3A1 (e), and proline (f), quantified by LC-MS/MS. Pearson correlation coefficients and p-values determined by two-tailed Student’s t-tests are displayed. g, Log2-transformed abundance of COL3A1 and proline in groups categorized by aortic degeneration (degenerated or healthy) quantified by LC-MS/MS. h, Log2-transformed abundance of proteins associated with aortic disease. Log2-transformed abundance of proteins associated with NAD+ metabolism with (i), or without (j), significant changes. k, Total levels of NAD+ and NADH (left), and the ratios of NAD+/NADH across patient subgroups: healthy (n = 10 patients), degenerated (n = 23 patients), degenerated with lower (n = 10 patients) or higher (n = 13 patients) aortic architecture score, measured by colorimetric assay. Related to Fig. 2. Data are represented as mean ± standard deviation, violin plots are presented as median and interquartile range, p-values were determined by Mann-Whitney U test or two-tailed Student’s t-tests.
Extended Data Fig. 4 Validation of SMC-specific deletion.
a, Aortas from control and smooth muscle-specific knockout of Nampt, Nmnat1, Slc25a51, Nadk2, Nmnat3 and Aldh18a1 mice were immunostained for NAMPT, NMNAT1, SLC25A51, NADK2, NMNAT3 and ALDH18A1, respectively (n = 3 male mice per group). Scale bar: 50 μm. Western blots of kidney (b), liver (c), heart (d), and aorta (n = 3 male mice per group) (e), tissues from control and smooth muscle-specific knockout mice for NAMPT, NMNAT1, SLC25A51, NADK2, NMNAT3 and ALDH18A1. The experiment was repeated three times with similar results (b, c, d).
Extended Data Fig. 5 Echocardiographic and electrocardiographic measurements of aortas in SMC-Nampt−/−, SMC-Nmnat1−/−, SMC-Slc25a51−/− and SMC-Nmnat3−/− mice, and the effect of Nmnat3 ablation on vascular pathology.
a, Schematic of the thoracic aorta showing measurement locations. b,c, Calculations of aortic pulse wave velocity and aortic strain. d, Representative aortic ultrasound images of 50 weeks old mice. Scale bar: 1 mm. e, Overview of the NAD+ salvage and transport pathway, and experimental timeline for arterial stiffening and aortic dilation analysis. f, Quantification of aortic diameter in 50 weeks old male mice, including Myh11-CreERT2 (n = 6), SMC-Nmnat3−/− (n = 9) and SMC-Slc25a51−/− (n = 10) mice. g, Pulse wave velocity and circumferential cyclic strain of the aorta of 50 weeks old male mice, including Myh11-CreERT2 (n = 6), SMC-Nmnat3−/− (n = 8) and SMC-Slc25a51−/− (n = 10). h, Representative histologic images showing HE and EVG section staining of ascending thoracic aorta. Scale bar: 100 μm and 50 μm. Data are represented as mean ± standard deviation, p-values were determined by two-tailed Studentʼs t-tests. The ultrasound data of Myh11-CreERT2 mice and SMC-Slc25a51−/− mice in Extended Data Fig. 5f, g, Extended Data Fig. 7b and main text Fig. 4d, e are identical (that is, representing the same data) to minimize the necessity for additional mice sacrifices.
Extended Data Fig. 6 Macroscopic images of aortas of mice infused with Ang II, and the effect of ablation of Nampt, Nmnat1, or Slc25a51 in smooth muscle cells on aortic NAD+ level and blood pressure.
a, Photographic images show whole aortas of mice that died from aortic rupture (right) and surviving mice euthanized at the end of 6-week experiment (left). Representative macroscopic images were shown in Fig. 5. Scale bar: 1 cm. b, Ablation of Nampt (n = 5), Nmnat1 (n = 5), or Slc25a51 (n = 5) in smooth muscle cells reduced aortic NAD+ level. c, Comparison of NAD+ levels in the skeletal muscle (n = 4), liver (n = 4) and heart (n = 4) tissues between 3-month-old and 12-month-old mice. d, Ablation of Nampt (n = 5), Nmnat1 (n = 5), or Slc25a51 (n = 5) in 40-week-old smooth muscle cell-specific knockout male mice did not alter diastolic or systolic blood pressure. Data are represented as mean ± standard deviation, p-values were determined by two-tailed Studentʼs t-tests. BP, blood pressure.
Extended Data Fig. 7 The effect of Nadk2 ablation on vascular pathology.
a, The experimental outline of spontaneous aortic dilation in mice. Ultrasonography was performed at 50 weeks of age. b, Representative aortic ultrasound images of 50 weeks old Myh11-CreERT2 (n = 6) and SMC-Nadk2−/− (n = 7) male mice. c, Representative aortic ultrasound images of 50 weeks old male mice. Scale bar: 1 mm. d, Representative histologic images showing HE and EVG section staining of ascending thoracic aorta. Scale bar: 100 μm and 50 μm. Data are represented as mean ± standard deviation, p-values were determined by two-tailed Studentʼs t-tests. The ultrasound data of Myh11-CreERT2 mice in Extended Data Fig. 5f, g, Extended Data Fig. 7b and main text Fig. 4d, e are identical (that is, representing the same data) to minimize the necessity for additional mice sacrifices.
Extended Data Fig. 8 Collagen and proline analysis in mouse aortas, and metabolic profiling in Slc25a51 knockdown cells.
a,c, Western blots and b,d, quantification analysis of collagen type III and type I in mouse aortas (n = 3 biological replicates). Samples with the same colored lines represent the same biological sample. e, Proline levels in mouse aorta, detected by LC-MS in 3-day Ang II-infused Myh11-CreERT2 (n = 4), SMC-Nampt−/− (n = 4), SMC-Nmnat1−/− (n = 4) and SMC-Slc25a51−/− (n = 4) mice. f, Low-magnification immunohistochemistry of type I and III collagen in mouse aortas corresponding to Fig. 6h. g, Low-magnification immunohistochemistry of type III collagen in aortas with proline supplementation corresponding to Fig. 7d (n = 3 biological replicates). Scale bar: 100 μm. h, Slc25a51 knockdown efficiency in A7R5 cells measured by RT-qPCR. i, Volcano plot of differentially expressed metabolites in Slc25a51 knockdown A7R5 cells. j, Heatmap of differentially expressed metabolites in Slc25a51 knockdown A7R5 cells. Data are represented as mean ± standard deviation, p-values were determined by two-tailed Studentʼs t-tests.
Extended Data Fig. 9 Infusion of Ang II predisposed SMC-Aldh18a1−/− mice to lethal aortic dissection and rupture.
a, Representative immunohistologic images showing the staining of type I and III collagens in mice aortas in 3-day Ang II infused mouse model (n = 3 biological replicates). Scale bar: 50 μm. b, The Experimental outline of 6-weeks Ang II infusion. Osmotic minipumps were implanted subcutaneously in SMC-Aldh18a1−/− mice male mice for continuous delivery of Ang II over a period of 6 weeks. c, Incidence and localization of lethal aortic dissections. Within 6 weeks of Ang II treatment, SMC-Aldh18a1−/− mice exhibits a mortality rate of 5/10. d, Representative macroscopic images of the whole aortas of mice that died from aortic rupture (second line) and euthanized mice at the end of experiment (first line) in each group. Postmortem examination reveals that all deceased mice exhibited aortic ruptures with accompanying hemothorax or hemoabdomen, while the surviving mice demonstrate aortic dilation and intramural hematoma. Scale bar: 1 cm. e, Representative histologic images showing HE and EVG staining of ascending thoracic aorta and abdominal aorta of surviving mice euthanized at the end of experiment (n = 3 biological replicates). Histological assessment of the aorta indicates hematomas, elastin lamina degradation, and medial thickening in the aortas of surviving mice. Scale bar: 100 μm and 50 μm.
Extended Data Fig. 10 Graphical representation illustrating the risk factors that influence vascular collagen homeostasis and contribute to vascular pathologies.
More severe vascular pathologies can be caused by well-recognized coding variations in genes associated with collagen production and TGFβ signaling. In contrast, common genetic variants of SLC25A51, by affecting type III collagen turnover, lead to slowly progressive vascular pathology, which worsen with concurrent conditions such as hypertension.
Supplementary information
Supplementary Tables
Supplementary Table 1. Clinical demographics of the patients included in histopathologic grading. Continuous variables are expressed as mean ± s.d. and compared using two-tailed Student’s t-test. Categorical variables are expressed as number (percentage) and compared using the χ2 test. ACEI/ARB, angiotensin-converting enzyme inhibitor/angiotensin receptor blocker. The grades used for the analysis involved grades 1 through 5, which correspond to the EVG histological stain and represent minimal to almost complete loss of elastic fibers. Aortic architecture scores of less than 3.4 were considered moderately degenerative, while scores above this threshold were classified as severely degenerative. Supplementary Table 2. Clinical demographics of all patients included in this study. Continuous variables are expressed as mean ± s.d. and compared using two-tailed Student’s t-test. Categorical variables are expressed as number (percentage) and compared using the χ2 test. Supplementary Table 3. Clinical demographics of the patients included in the proteome analysis. Continuous variables are expressed as mean ± s.d. and compared using two-tailed Student’s t-test. Categorical variables are expressed as number (percentage) and compared using the χ2 test. Supplementary Table 4. Clinical demographics of the patients included in the metabolome analysis. Continuous variables are expressed as mean ± s.d. and compared using two-tailed Student’s t-test. Categorical variables are expressed as number (percentage) and compared using the χ2 test. Supplementary Table 5. Clinical demographics of the patients included in the transcriptome analysis. Continuous variables are expressed as mean ± s.d. and compared using two-tailed Student’s t-test. Categorical variables are expressed as number (percentage) and compared using the χ2 test. Supplementary Table 6. Clinical and histological characteristics of patients. Supplementary Table 7. Numbering and grouping of the proteome. Supplementary Table 8. Differentially expressed proteins in diseased versus healthy groups. P values were determined by Mann–Whitney U-test; the Benjamini–Hochberg method was used to correct for multiple comparisons (threshold, P < 0.05). Supplementary Table 9. Differentially expressed genes in diseased versus healthy groups. P values were determined by limma statistical analysis; the Benjamini–Hochberg method was used to correct for multiple comparisons (threshold, P value < 0.05). Supplementary Table 10. Thoracic aortic metabolome from 73 patients and differentially expressed metabolites in diseased versus healthy groups. For metabolites with the same ID in positive and negative ion modes, metabolites with higher intensity were retained. Orthogonal partial least squares discrimination analysis was used to identify differentially expressed metabolites (variable importance in projection > 1). P values were determined by limma statistical analysis; the Benjamini–Hochberg method was used to correct for multiple comparisons (threshold, P value < 0.05). Supplementary Table 11. Differentially expressed proteins in higher versus lower aortic architecture score groups. P values were determined by Mann–Whitney U-test; the Benjamini–Hochberg method was used to correct for multiple comparisons (threshold, P value < 0.05). Supplementary Table 12. Differentially expressed genes in higher versus lower aortic architecture score groups. P values were determined by limma statistical analysis; the Benjamini–Hochberg method was used to correct for multiple comparisons (threshold, P value < 0.05). Supplementary Table 13. Correlation between aortic architecture scores and protein abundance. Pearson correlation coefficients and P values determined by two-tailed Student’s t-tests are displayed. Supplementary Table 14. Mouse aortic proteome. Proteome from 3-d Ang II-induced aortic ECM remodeling mouse experiments generated with a DDA approach. Supplementary Table 15. Differentially expressed proteins in 3-d Ang II-infused control compared to saline-infused control. P values were determined by two-tailed Studentʼs t-tests; the Benjamini–Hochberg method was used to correct for multiple comparisons (threshold, P < 0.05). Supplementary Table 16. Differentially expressed proteins in 3-d Ang II-infused SMC-Slc25a51−/− mice compared to Ang II-infused controls. P values were determined by two-tailed Studentʼs t-tests; the Benjamini–Hochberg method was used to correct for multiple comparisons (threshold, P < 0.05). Supplementary Table 17. Differentially expressed proteins in 3-d Ang II-infused SMC-Nadk2−/− mice compared to Ang II-infused controls. P values were determined by two-tailed Studentʼs t-tests; the Benjamini–Hochberg method was used to correct for multiple comparisons (threshold, P < 0.05). Supplementary Table 18. Transcriptome-wide association analysis results for aortic aneurysm/dissection and five genes related to NAD+ salvage and mitochondrial transport. P values were calculated using SMR analysis, and the Benjamini–Hochberg method was applied to correct for multiple comparisons.
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Zhang, J., Tang, Y., Zhang, S. et al. Mitochondrial NAD+ deficiency in vascular smooth muscle impairs collagen III turnover to trigger thoracic and abdominal aortic aneurysm. Nat Cardiovasc Res 4, 275–292 (2025). https://doi.org/10.1038/s44161-024-00606-w
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DOI: https://doi.org/10.1038/s44161-024-00606-w
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Mitochondrial NAD+ transporter SLC25A51 linked to human aortic disease
Nature Cardiovascular Research (2025)
