Deciphering the role of cuproptosis in the development of intimal hyperplasia in rat carotid arteries using single-cell analysis and machine-learning techniques

Objective: This study aims to explore the regulatory role of cuproptosis in carotid intimal hyperplasia (IH), providing new insights into its pathophysiological mechanisms and potential diagnostic and therapeutic strategies. Methods: We downloaded single-cell sequencing and bulk transcriptome data from the GEO database to screen for copper-growth-associated genes (CAGs) using machine-learning algorithms, including Random Forest and Support Vector Machine. After identifying relevant genes, we verified CAGs expression in IH and control groups using a rat model of carotid balloon strain. We analyzed the immune infiltration characteristics of carotid intimal hyperplasia and used electron microscopy to observe mitochondrial structural changes in cuproptosis. Additionally, we performed subgroup analyses of carotid balloon strains. The cuproptosis activity of VSMCs was explored in a single-cell dataset. Immunohistochemistry was applied to validate the expression of CAGs. Results: By means of machine learning algorithms, we identified several genes, including Pdhx and Fdx1, as novel therapeutic targets for carotid intimal hyperplasia. Meanwhile, immunohistochemistry results observed decreased expression of Pdhx and Fdx1 in the Neointimal hyperplasia(Neo) group. Immunohistochemical results showed a difference in cellular infiltration between Dendritic cells resting and Mast cells resting. By calculating cuproptosis activity in vascular smooth muscle cells (VSMCs), we found increased cuproptosis activity in normal vascular smooth muscle cells which was also observed in the electron microscopy. Microscopy revealed less mitochondrial swelling characteristic of cuproptosis in Neo group. Conclusion: The CAGs identified may regulate intimal hyperplasia in rat carotid arteries by modulating cuproptosis and represent potential targets for treatment.