Proteome Differences in Smooth Muscle Cells from Diabetic and Non-Diabetic Abdominal Aortic Aneurysm Patients Reveal Metformin-Induced Mechanisms

Aims: Surgery remains the only definitive treatment option for abdominal aortic aneurysms (AAA), as no conclusive evidence supports drug effectiveness in preventing AAA growth. Although type 2 diabetes (T2D) is an important cardiovascular risk factor, patients with T2D show reduced AAA presence and growth, associated with metformin use. We aimed to investigate the potential benefits of metformin on AAA using proteomics and in vitro experiments. Methods: Proteomics analysis using tandem mass spectrometry was performed on aortic smooth muscle cells (SMCs) from non-pathological controls (C-SMC, n = 8), non-diabetic (ND, n = 19) and diabetic (D, n = 5) AAA patients. Key findings were subsequently validated in aortic tissue using mass spectrometry-based proteomics. SMCs were cultured with/without metformin and analyzed. Results: Comparison of the proteome of SMCs from ND-AAA patients with controls revealed a reduction in proteins associated with metabolic processes and mitochondrial function. Cytoskeletal and extracellular matrix (ECM) proteins were elevated in ND-AAA-SMCs versus C-SMCs, with a similar cluster of mechanosensitive proteins being increased in ND-AAA-SMCs versus D-AAA-SMCs. D-AAA-SMCs showed an improved metabolic and antioxidant profile, enriched in pentose phosphate pathway proteins responsible for NAD(P)H generation (G6PD, PGD) and NAD(P)H-dependent antioxidants (NQO1, CBR1, AKR1C1, AKR1B1, GSTM1), all regulated by NRF2, an antioxidant transcription factor. Over half of the proteins identified in the protein–protein interaction network, constructed from proteins with higher expression in D-AAA SMCs versus ND-AAA SMCs, were verified in D-AAA aortic tissue. In vitro, metformin causes a shift from aerobic to anaerobic metabolism, increased AMPK activation and elevated mitochondrial biogenesis, indicated by increased PGC-1α expression. Metformin increased the gene expression of PGD, CBR1 and the protein expression of NQO1, with enhanced translocation of pNRF2 to the nucleus, due to reduced KEAP1 as negative regulator of NRF2. Consequently, metformin enhanced the gene expression of well-known antioxidant regulators SOD2 and CAT. Conclusions: This study identified significant differences in the proteome of SMCs derived from controls, ND-AAA and D-AAA patients. It highlights distinct pathways in relation to mechanosensing, metabolism and redox balance as therapeutic targets of metformin that may underlie its inhibition of AAA progression.