Background
Coronary artery disease (CAD) and acute coronary syndrome (ACS) remain leading causes of morbidity and mortality worldwide. Endothelial dysfunction and inflammation are key factors in disease progression. While the impact of the individual genetic background is appreciated, it remains difficult to clearly assess its impact as a disease modifier. Induced pluripotent stem cells (iPSCs) are an ideal tool as they retain donor genetics while largely eliminating age- and lifestyle-related epigenetic changes. To address the gaps in the current models, we utilized iPSC-derived endothelial cells (iPSC-ECs) to study the cellular mechanisms underlying endothelial dysfunction in ACS patients.
Aim
We aimed to characterize differences in endothelial function, such as cell growth, inflammatory activation and senescence, between ACS patients and healthy individuals using a patient-specific iPSC-EC model.
Methods
iPSC-ECs were differentiated from healthy controls and young (<65 years old) ACS patients. We characterized these cells using several techniques: DNA-methylation analysis (Illumina EPIC chip) to assess mitotic age, RNA-sequencing (RNA-seq) for gene expression differences and pathway analysis (GSEA), and flow cytometry for adhesion molecule expression (E-Selectin, VCAM-1, ICAM-1). Functional assays included a proliferation assay, tube formation assay, scratch wound healing assay and nitric oxide (NO) production measurments. Senescence was assessed via qPCR (Sirt1, Sirt3, p21), Western blot (Sirt1), and senscence-associated beta galactosidase (SA-b-gal) staining. Mitochondrial and metabolic function was assessed with Seahorse Flux analysis (Mito Stress Test, Glycolysis Stress Test) as was mitophagy via a mitophagy dye assay. Finally, we tested the effects of direct and indirect Sirt1 activation using Metformin and SRT1720, a specific Sirt1 activator.
Results
ACS iPSC-ECs exhibited an RNA-seq profile enriched for senescence, autophagy, and DNA damage pathways. This was accompanied by enhanced adhesion molecule expression, particularly under inflammatory conditions. Functionally, ACS iPSC-ECs showed slower growth and impaired wound healing, alongside elevated molecular markers of senescence, including higher p21 mRNA and SA-β-gal and lower Sirt1 protein expression as well as lower NO prodution. Metabolically, ACS iPSC-ECs showed lower ATP production from oxidative respiration and a compensatory higher ATP production from glycolysis. This metabolic shift, also known as Warburg effect, was accompanied by impaired mitophagy. Crucially, treatment with Metformin and SRT1720 partially rescued the senescent and dysfunctional phenotype, improving key metabolic and functional readouts.
Conclusion
Taken together, ACS iPSC-ECs exhibit a distinct, patient-specific premature-senescence phenotype with metabolic and mitochondrial dysfunction. This model identifies cellular senescence and its associated pathways, particularly the loss of Sirt1-mediated functions and impaired mitophagy, as potential drivers of persistent endothelial dysfunction in ACS patients. Our findings suggest that targeting these pathways with metabolic modulators like dirct and indirect Sirt1 activators represents a promising therapeutic strategy for improving vascular health in ACS patients.
