Gut Microbiota-Dependent Increase in Phenylacetic Acid Induces Endothelial Cell Senescence During Aging

Geriatrics Microbiology Cell Biology Gastroenterology Biochemistry Cardiovascular System Life Sciences Medicine
gut microbiota phenylacetic acid endothelial cell senescence aging mechanisms cardiovascular disease oxidative stress

Molecular Mechanisms of Gut Microbiota and Its Metabolites Mediating Endothelial Cell Senescence During Aging – Interpretation of Latest Results from Nature Aging

I. Research Background and Significance

Against the backdrop of a deeply aging human society, cardiovascular disease (CVD) has become one of the greatest health threats to the global elderly population. Epidemiological evidence shows that aging is one of the most important risk factors for CVD. However, the key cellular basis leading to the functional decline of the cardiovascular system—endothelial cell (EC) dysfunction, especially EC senescence—still lacks full elucidation regarding its molecular triggers and regulatory mechanisms in vivo.

In recent years, gut microbiota, known as the “second human genome after the genome itself,” have gained recognition in medical and life sciences as being closely linked to host metabolism, immunity, inflammation, and various systemic disease states. A growing body of evidence suggests that gut microbiota, by breaking down dietary components, produce a range of metabolites—including beneficial short-chain fatty acids (SCFA) and potentially harmful aromatic amino acid derivatives (such as phenylacetic acid, PAA). These compounds can impact cardiovascular health, inflammation, and even the aging process via the gut–vascular axis.

Notably, preclinical and clinical studies indicate that certain gut microbes metabolize dietary phenylalanine to generate PAA and its downstream product, phenylacetylglutamine (PAGln). The increased levels of these metabolites are associated with adverse cardiovascular events and all-cause mortality in patients with anemia or chronic kidney disease. However, how PAA acts on vascular endothelial cells to promote their aging, trigger vascular dysfunction, and the underlying molecular networks remain insufficiently explored.

This study was carried out precisely against such clinical needs and scientific gaps, aiming to reveal the novel “gut microbiota–phenylacetic acid–vascular endothelial senescence” axis and to further explore the intervention potential of SCFA (especially acetate) in anti-endothelial senescence and vascular health maintenance. This can provide new ideas and targets for the prevention and treatment of aging-related cardiovascular diseases.

II. Source of the Paper and Author Information

This research paper, titled “Gut microbiota-dependent increase in phenylacetic acid induces endothelial cell senescence during aging,” was published in the June 2025 issue of Nature Aging (Nature Aging, Volume 5, June 2025, Pages 1025–1045), DOI: https://doi.org/10.1038/s43587-025-00864-8.

The study was jointly conducted by Seyed Soheil Saeedi Saravi (corresponding author), Benoit Pugin, Florentin Constancias, Khatereh Shabanian, and several others, with the research team coming from top international institutions such as the University of Zurich, École Polytechnique Fédérale de Lausanne, St. Gallen Hospital, University of Bergamo (Italy), Imperial College London, and University Hospital Zurich. The work demonstrates a multidisciplinary and international collaboration, characterized by high innovation and authority.

III. Research Design and Experimental Process

1. Overall Research Framework

This research centers on “how the gut microbiota metabolite PAA promotes vascular endothelial cell senescence and the intervention potential of acetate in this process.” The study establishes a complete research chain from large clinical cohorts to animal models, then to cellular and molecular mechanisms. The research process mainly includes:

  • Clinical data analysis of a large human population cohort (TwinsUK cohort, n=7303)
  • Animal models (mice) and studies on aging
  • Integrated analysis of gut microbiome and metabolomics
  • Functional validation of specific bacterial species (Clostridium sp. asf356)
  • In situ and in vitro EC phenotyping and mechanistic experiments
  • Innovative intervention studies (acetate supplementation, combined senolytic drugs)

2. Specific Experimental Steps and Methods

2.1 Dynamics of PAA and PAGln Levels in Clinical Cohorts and Animal Models

  • TwinsUK Human Cohort: Non-targeted metabolomics was used to measure plasma PAA and PAGln levels in 7,303 healthy subjects aged 18-95. Linear regression and correlation analyses clarified the relationship between these metabolites and age.
  • Mouse Animal Model: Selected 3-month-old (young) and 24-month-old (aged) C57BL/6J mice. Targeted metabolomics collected plasma PAA and PAGln levels, with sample size n=6. All mice were matched for sex, weight, and renal function to exclude renal confounding.

2.2 Gut Microbiome Analysis and Identification of PAA-Synthesizing Pathways

  • Mouse Fecal Shotgun Metagenomic Sequencing: Fresh fecal samples from mice of various ages were subjected to shotgun metagenomic sequencing.
  • KEGG Database Pathway Alignment: Focused on two oxidative decarboxylation pathways key to PAA synthesis: phenylpyruvate:ferredoxin oxidoreductase (PPFOR) and α-ketoisovalerate:ferredoxin oxidoreductase (VOR). Analyzed gene abundance changes in different age groups.
  • ANCOM Algorithm and CLR Transformation: Identified differentially abundant bacterial community structures, particularly the change in PPFOR/VOR-positive functional strains.

2.3 Functional Verification of Associated Specific Bacterial Species

  • Species Correlation Analysis: Spearman rank correlation analysis was used to identify dominant bacteria positively associated with plasma PAA.
  • In Vitro Functional Culture Experiments: Clostridium sp. asf356 was cultured in media containing phenylalanine, with LC-MS/MS detecting its PAA and PAGln production.
  • Retrospective Analysis of TwinsUK Microbiome Data: Meta-taxonomic validation was conducted in 900 cohort samples, confirming the association of PPFOR-positive Enterobacteria with PAA/PAGln.

2.4 Validation of Physiological and EC Senescence Phenotypes in Animal Models

  • Clostridium sp. asf356 Colonization Experiments: 10-12-week-old antibiotic-pretreated mice were colonized with this strain via oral administration. Plasma PAA levels, arterial elasticity (force tension), and EC senescence markers (SA-β-Gal staining, p16^Ink4a, DNA damage marker γ-H2A.X, inflammatory cytokines IL-1β/IL-6) were compared between groups.
  • Senolytic Drug Intervention: Combined dasatinib (5mg/kg) and quercetin (50mg/kg) were used to treat the colonized group to clear senescent cells, examining whether arterial function and EC angiogenic capability could be restored.

2.5 In Vivo and In Vitro Validation of Direct PAA Mechanisms

  • In Vitro Human Aortic ECs (HAEC): Treated under serum-free conditions with 10μM PAA for 72 hours. Compared to replicative senescence controls (Passage 15-17), assessed for SA-β-Gal, Ki67, tumor suppressor genes (p16Ink4a, etc.), inflammatory cytokines, and VCAM1 phenotypes.
  • In Vivo PAA Infusion Experiments: Four-week intraperitoneal PAA treatment in mice; assessed vascular structure (elasticity, type III collagen, MMP-9), EC senescence/DNA damage/inflammatory phenotype, and microangiogenesis.
  • Mito-HyPer7.2 Biosensor Innovation: Adenoviral transduction of Hyper7.2 targeting mitochondria to dynamically detect PAA-induced mitochondrial H₂O₂ production.
  • Seahorse XF Analyzer: Monitored baseline and maximal respiration (OCR), glycolysis (ECAR), and ATP generation in real-time, evaluating PAA’s impact on EC energy metabolism.

2.6 Exploration of Acetate’s Anti-Senescence Potential

  • In Vitro Experiments of Acetate Plus PAA: Supplemented with 3μM sodium acetate, evaluated its antagonism of PAA-induced EC senescence, ROS, energy impairment, SASP, telomere maintenance (hTERT), and DNA damage.
  • Molecular Mechanism Exploration: Investigated acetate’s regulatory effects on the NAD+–SIRT1–NRF2 antioxidant pathway and NF-κB inflammatory pathway, with loss-of-function experiments (si-SIRT1, NRF2 inhibitor) to validate causality.

IV. Key Experimental Results and Data at Each Stage

1. Dynamic Changes of PAA and PAGln in Human and Animal Models

  • Plasma levels of PAA (r=0.06, p<0.001) and PAGln (r=0.25, p<0.001) significantly increased with age; this phenomenon was independent of renal function.
  • Increased PAA and PAGln were also observed in aged mice, confirming tight association with the aging process.

2. Microbiome Functional Pathway Shifts and Specific Bacterial Enrichment

  • Metagenomic functional analysis showed a highly significant increase (p<1e-4) in PPFOR/VOR gene abundance in feces of aged mice. The ratio of PPFOR+ and VOR+ strains in the aged group was 72%, much higher than the young group (42%).
  • Clostridium sp. asf356 was the only PPFOR+ species significantly associated with increased PAA, with a notable rise in its relative abundance in aged mouse microbiota.
  • The TwinsUK cohort also verified a significant positive correlation between Clostridium (PPFOR+ gene) and PAA levels (p=2.45e-5).

3. Clostridium sp. asf356 Colonization Experiments

  • Colonization with this bacterium raised plasma PAA by approximately 3.15 times and PAGln by about 1.7 times, reduced arterial elasticity, increased perivascular and visceral fat, comprehensive elevation in senescence, DNA damage, inflammation markers (p16Ink4a, γ-H2A.X, IL-1β/IL-6, SA-β-Gal), and significantly diminished vascular relaxation and angiogenic ability.
  • Combined senolytic drugs effectively eliminated these senescent phenotypes and restored EC function.

4. In Vivo and In Vitro Studies of PAA-Induced Premature Senescence

  • PAA directly induced human EC phenotypes highly similar to replicative senescent cells: cells became large, flat, multinucleated; Ki67 decreased; p16Ink4a/p19Ink4d/p21 upregulated; SASP secretion (IL-1β/IL-6/VCAM1, etc.) enhanced; DNA damage was evident.
  • Dasatinib and quercetin could selectively clear PAA-induced senescent cells and significantly restore angiogenic ability.

5. Analysis of PAA-Induced ROS, Energy Impairment, and Epigenetic Regulatory Networks

  • PAA prompted excessive mitochondrial H₂O₂ production in ECs (Mito-Hyper7.2 redox ratio more than doubled), upregulation of NADPH oxidase NOX4, and downregulation of antioxidant defense gene GPX1, exacerbating oxidative stress.
  • Seahorse analysis showed baseline respiration, maximal respiration, and ATP generation all plummeted in the PAA group (up to 40–50% drop); glycolytic capacity also declined significantly, indicating impaired energy metabolism.
  • At the epigenetic level, PAA mediated H₂O₂-induced CaMKII phosphorylation, promoted HDAC4 phosphorylation and cytoplasmic translocation, removed inhibition on key inflammatory SASP genes (like VCAM1), suppressed eNOS phosphorylation, and impaired vascular relaxation and angiogenesis.

6. Acetate Protective Intervention and Signaling Pathway Mechanism

  • Fecal acetate in aged mice dropped by 80% compared to young mice, correlating with reduced probiotics (Prevotella, Rikenellaceae).
  • Acetate supplementation markedly reduced PAA-induced senescence phenotypes (Saβ-Gal, p16Ink4a, γ-H2A.X), boosted hTERT, reversed telomere shortening and DNA damage.
  • Sodium acetate significantly improved energy metabolism (OCR up 30–40%), elevated NAD+ levels, activated NAD+-dependent deacetylase SIRT1, upregulated NRF2—GPX1/PRDX3 and other antioxidant enzymes, suppressed NF-κB-mediated SASP secretion, and dramatically restored angiogenic capability.
  • siRNA experiments clarified that acetate’s anti-senescence effects are regulated via the SIRT1–NRF2 and SIRT1–NF-κB pathways; pharmacological inhibitor studies further substantiated the dual role of energy–antioxidant metabolic regulation.

V. Research Conclusions, Value, and Highlights

1. Main Conclusions

This study for the first time reveals that gut microbiota-derived PAA serves as a key molecule driving endothelial senescence during aging, acting through NOX4-mediated excessive mitochondrial H₂O₂ generation, activating the SASP phenotype, epigenetic regulation (HDAC4/VCAM1/eNOS modification), and energy impairment, eventually resulting in vascular dysfunction and reduced angiogenesis. It first identifies Clostridium sp. asf356 as the key species driving this process, providing a target for future precise microbiome interventions. Simultaneously, it systematically validates that acetate supplementation can significantly curb PAA-induced EC senescence phenotypes through the SIRT1/NRF2 antioxidant-energy and SIRT1–NF-κB inflammation-suppression pathways, establishing acetate as an efficient microbial senomorphic intervention strategy.

2. Scientific Significance and Application Value

  • Reveals the causal link between aging-related microbiota–metabolite–vascular dysfunction, deepening the molecular map of the gut–vascular aging axis.
  • Proposes PAA and its functional bacteria as biomarkers and intervention targets for vascular aging and atherosclerosis, providing a basis for precise intervention.
  • Clarifies the therapeutic potential of acetate, a key SCFA produced by a healthy gut microbiota, in anti-EC senescence and functional restoration, supporting the development of SCFA supplements, electrophysiological modulation, and other novel prevention and treatment strategies.

3. Research Highlights and Innovations

  • Integrates multi-omics (metagenomics + metabolomics), large human cohorts, and molecular phenotyping with a systematic and rigorous research process.
  • Innovatively mines and verifies the PAA-induced multi-layer signal network of “energy+epigenetics+oxidative stress+SASP,” proposing a new idea for senomorphic intervention.
  • Employs advanced techniques such as Mito-Hyper7.2 real-time redox sensing, promoting high spatiotemporal resolution mechanistic analysis of cellular oxidative stress events.

VI. Other Valuable Content and Prospects

  • The study also evaluates the effects of senolytic drugs (Dasatinib+Quercetin) in the model of microbe-derived metabolite-induced senescence, providing experience for drug–microbiota synergistic interventions.
  • Suggests that gut microbiome restoration (e.g., promoting microbiota diversity, supplementing acetate-producing bacteria) may develop into a core strategy for delaying cardiovascular aging, especially in the elderly/high-risk populations.
  • Has theoretical demonstration value for future basic and translational research based on functional microbiota genomics, “transplant + ecobiotic + nutritional intervention” approaches.

VII. Summary

This study unifies evidence from clinical and basic research, establishing for the first time a complete mechanistic pathway of “gut microbiota–metabolite PAA–endothelial senescence–functional impairment,” and provides theoretical foundations and novel intervention targets for atherosclerosis and aging-related cardiovascular disease prevention and control. Microbial senomorphics (such as acetate) may usher in a new chapter of healthy aging, offering systemic solutions for managing cardiovascular health in the global elderly population.