Metabolomic response to metformin therapy in experimental abdominal aortic aneurysm

1. Overview

Abdominal aortic aneurysm (AAA) is an age-associated degenerative disorder marked by progressive dilation of the aorta, particularly within the infrarenal segment, as a result of matrix metalloproteinase-mediated extracellular matrix degradation. This condition predominantly affects individuals over the age of 60 years. To date, there are no FDA-approved pharmacological interventions that reliably prevent AAA development or effectively halt disease progression; consequently, surgical repair remains the only available therapeutic option for patients with AAA. Recent research by our group and others has demonstrated that the anti-diabetic medication metformin attenuates experimental AAAs and correlates with decreased prevalence and slowed progression of clinical AAAs^1-4. Ongoing clinical trials are evaluating metformin’s therapeutic potential in limiting AAA advancement⁵, yet the underlying mechanisms remain unclear. Given metformin’s primary effects on metabolic and inflammatory pathways, we hypothesize that its ability to ameliorate AAA disease may partially stem from reprogramming metabolic processes.

The proposed study has received approval from the Stanford University IACUC. Experiment preparations will be undertaken in March 2026, with experiments commencing on April 1, 2026, and continuing through September 30. Data analysis and preparation of research reports, abstracts, and manuscripts are scheduled for October 2026. Experimental findings will be disseminated within the scientific community via presentations at academic conferences and subsequent journal submissions. Raw data will be securely stored in the unlimited Stanford Medicine Box and made accessible to qualified researchers upon reasonable request.

2. Introduction

AAA is defined histopathologically by persistent inflammation, extracellular matrix breakdown, vascular smooth muscle cell depletion, and remodeling of the medial layer.

Metformin, a primary anti-diabetic agent, has been observed in our research and corroborated by other studies to correlate with decreased prevalence and slower progression of both clinical and experimental AAA^1-4,6. Treatment with metformin mitigates critical pathological aspects of AAA, including medial elastin and smooth muscle destruction, leukocyte infiltration, and neoangiogenesis, partially through activation of AMP-activated protein kinase^1,7. Multiple ongoing clinical trials are investigating the therapeutic potential of metformin in curbing AAA progression⁵. Although metformin demonstrates promising effects in experimental and potentially clinical AAAs, the metabolomic pathways responsible for these protective outcomes have yet to be fully clarified.

We hypothesize that metformin attenuates experimental AAA progression by altering metabolic pathways associated with inflammation. This proposal aims to address this hypothesis through two objectives:

· To evaluate the metabolomic response to metformin therapy in experimental AAA models.

· To identify principal metabolic pathways and biomarkers linked to metformin-mediated AAA suppression.

Examining the effects of metformin on the metabolome in AAA will enhance understanding of disease-associated pathways, facilitate the detection of circulating biomarkers, and offer mechanistic rationale for drug repurposing. This investigation may contribute to the development of metabolism-driven therapeutic approaches for AAA, which currently lacks effective pharmacological treatments. Furthermore, it will provide both technical and scientific guidance for comprehensive, explosive analyses of metabolomic responses and therapeutic mechanisms among AAA patients enrolled in the LIMIT trial.

3. Methods

Participants/Sample

This experimental study will include serum, aneurysmal aorta, liver, and small intestine samples from all mice for metabolomic analysis, excluding those that die due to unforeseen circumstances. According to prior research, a sample size of ten mice is expected to provide adequate statistical power for subsequent analyses.

Materials and Procedures

Induction of Experimental AAAs: Abdominal aortic aneurysms (AAAs) will be induced in male C57BL/6J mice aged 10 to 12 weeks (The Jackson Laboratory, Bar Harbor, ME) through transient intra-aortic infusion of type I porcine pancreatic elastase (PPE). All surgical procedures will be performed under anesthesia (2% isoflurane inhalation) using aseptic techniques⁷. The infrarenal aorta will be exposed via an abdominal midline laparotomy. All visible aortic branches within the infrarenal segment will be ligated using 10-0 silk suture, and temporary occlusion of aortic blood flow will be achieved with peri-aortic circumferential 6-0 silk sutures. Aortotomy will be created with a 30-gauge needle in the designated segment, followed by infusion of 30 μL PPE through extruded polyethylene tubing for five minutes under constant pressure. Upon completion of the infusion, the aortotomy and laparotomy will be sequentially closed with 10-0 and 6-0 sutures, respectively. Postoperatively, mice will recover and be housed individually with unrestricted access to food and water for subsequent evaluations.

Metformin treatment: Three days post-PPE infusion, at which point all mice are expected to develop AAA (defined as greater than 50% increase in aortic diameter from baseline), mice will receive daily treatment with metformin hydrochloride (250 mg/kg; oral gavage) prepared in saline, or saline alone as vehicle control (n=10 mice per group)⁷. The selected metformin dose (250 mg/kg in mice) is equivalent to the maximum clinical dosage (2500 mg/day for a 70 kg patient) recommended for type 2 diabetes when scaled allometrically.

Sample collection: Fourteen days following PPE infusion, mice will be euthanized via carbon dioxide inhalation. Blood samples will be collected via cardiac puncture for serum preparation. Subsequently, mice will be perfused through the left ventricle, and tissues including the aneurysmal aorta, liver, and small intestine will be harvested and stored at -80°C for metabolite extraction.

Metabolite data acquisition: To profile changes in metabolites following metformin therapy, untargeted liquid chromatography–mass spectrometry (LC-MS/MS (tandem MS)) will be performed^8-10. Metabolites will be extracted using methanol (serum) or acetonitrile (aorta, liver and small intestine), and data acquisition will be performed on the Agilent 1290 Infinity LC system coupled with Agilent TOF 6545 utilizing a C18 reverse phase column and operated in both positive and negative electrospray ionization modes.

Planned Analyses

Data processing and statistical analyses will be conducted utilizing several bioinformatics tools, including the R program (version 4.2.1), MSConvert (Version 2.1), MetaboAnalystR database (Version 6.0), and the KEGG database, consistent with our prior and preliminary studies (refer to Preliminary data)^8-10.

Principal component analysis (PCA) will be employed to visualize metabolite distribution and correlations across individual samples, interventions (metformin versus vehicle), and cross-tissue/compartment comparisons. Additional analyses such as partial least squares-discriminant analysis (PLS-DA), volcano plots, and pathway enrichment methods will be used to further characterize and enhance the differentiation of the metabolome between intervention groups and among organs/compartments, thereby identifying the most significantly affected pathways and metabolites.

Comparative analyses will be performed on aneurysmal aorta (lesion), serum (systemic), liver (one key metformin target site), and small intestine (another key metformin target organ) to discern common and tissue-specific changes following metformin treatment. When needed, subsequent validation experiments will involve HPLC analysis to identify and quantify the top 10 significantly altered, both increased and decreased, metabolites potentially related to AAA pathogenesis.

Based on our preliminary data, it is anticipated that metformin therapy will induce tissue-specific metabolomic alterations, particularly affecting glycolysis, lipid metabolism and arachidonic acid metabolism. Additionally, metformin may restore mitochondrial-related metabolite profiles, attenuate pro-inflammatory lipid mediators, and normalize redox metabolites.

Ethics and Data Management

The proposed experiments have received approval from the IACUC at Stanford University. Experimental data will be disseminated to the scientific community through presentations at national or international conferences, followed by submission for journal publication. Raw data will be securely stored in the unlimited Stanford Medicine Box and made available to qualified researchers upon reasonable request.

4. Pilot Data (Optional)

In preliminary studies, we established experimental abdominal aortic aneurysm (AAA) models: PPE infusion in wild-type male C57BL/6J mice (Figure panels A-C) and chronic angiotensin II (Ang II) infusion in hyperlipidemic ApoE deficient mice (Figure panel A). Distinct metabolomic profiles were observed between AAA and non-AAA mice in both AAA models (Figure panel A). Administration of metformin in mice with existing AAA induced by PPE infusion resulted in alterations to these metabolomic profiles (panel A), with several metabolites showing increased or decreased levels compared to vehicle treatment. The preliminary data confirms the feasibility of the methodology and highlights the significance of metabolomic changes in response to metformin therapy.

5. Budget

Mouse purchase and housing: $3000.00; Surgical supplies for AAA model: $500.00; Supplies and instrument use charge: $5000.00; Metformin and other reagents: $1000.00; other cost: $500.00.

6. References

1 Fujimura, N. et al. Metformin treatment status and abdominal aortic aneurysm disease progression. J Vasc Surg 64, 46-54.e48, doi:10.1016/j.jvs.2016.02.020 (2016).

2 Golledge, J. et al. Editor's Choice - Metformin Prescription is Associated with a Reduction in the Combined Incidence of Surgical Repair and Rupture Related Mortality in Patients with Abdominal Aortic Aneurysm. Eur J Vasc Endovasc Surg 57, 94-101, doi:10.1016/j.ejvs.2018.07.035 (2019).

3 Golledge, J. et al. Association between metformin prescription and growth rates of abdominal aortic aneurysms. Br J Surg 104, 1486-1493, doi:10.1002/bjs.10587 (2017).

4 Itoga, N. K. et al. Metformin prescription status and abdominal aortic aneurysm disease progression in the U.S. veteran population. J Vasc Surg 69, 710-716.e713, doi:10.1016/j.jvs.2018.06.194 (2019).

5 Wanhainen, A. & Dalman, R. L. Update on Ongoing Randomised Controlled Trials Evaluating the Protective Effect of Metformin on Abdominal Aortic Aneurysm Progression. European Journal of Vascular and Endovascular Surgery 69, 6-8, doi:https://doi.org/10.1016/j.ejvs.2024.08.042 (2025).

6 Yu, X. et al. Metformin prescription and aortic aneurysm: systematic review and meta-analysis. Heart 105, 1351-1357, doi:10.1136/heartjnl-2018-314639 (2019).

7 Xu, B. et al. Mechanisms and efficacy of metformin-mediated suppression of established experimental abdominal aortic aneurysms. JVS Vasc Sci 4, 100102, doi:10.1016/j.jvssci.2023.100102 (2023).

8 Ye, J. et al. Metabolomics-Guided Hypothesis Generation for Mechanisms of Intestinal Protection by Live Biotherapeutic Products. Biomolecules 11, doi:10.3390/biom11050738 (2021).

9 Ye, J. et al. Reduced Sulfur Diet Reshapes the Microbiome and Metabolome in Mild-Moderate Ulcerative Colitis. Int J Mol Sci 26, doi:10.3390/ijms26104596 (2025).

10 Kulkarni, C. et al. A fasting-mimicking diet in patients with mild-to-moderate Crohn's disease: a randomized controlled trial. Nat Med, doi:10.1038/s41591-025-04173-w (2026).

Metabolomic response to metformin therapy in experimental abdominal aortic aneurysm

Funding Progress

Nonprofit Recipient

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Funding Progress

Nonprofit Recipient

Topics

DOI

Metabolomic response to metformin therapy in experimental abdominal aortic aneurysm

Funding Progress

Nonprofit Recipient

Topics

DOI