Differential Gene Expression and Immune Cell Infiltration in Carotid Intraplaque Hemorrhage Identified Using Integrated Bioinformatics Analysis

BACKGROUND: Intraplaque hemorrhage (IPH) is an important feature of unstable plaques and an independent risk factor for cardiovascular events. However, the molecular mechanisms contributing to IPH are incompletely characterized. We aimed to identify novel biomarkers and interventional targets for IPH and to characterize the role of immune cells in IPH pathogenesis. METHODS: The microarray dataset GSE163154 which contain IPH and non-IPH plaque samples was obtained from the Gene Expression Omnibus (GEO). R software was adopted for identifying differentially expressed genes (DEGs) and conducting functional investigation. The hub genes were carried by protein-protein interaction (PPI) network and were validated by the GSE120521 dataset. CIBERSORT deconvolution was used to determine differential immune cell infiltration and the relationship of immune cells and hub genes. We confirmed expression of proteins encoded by the hub genes by immunohistochemistry and western blotting in 8 human carotid endarterectomy samples with IPH and 8 samples without IPH (non-IPH). RESULTS: We detected a total of 438 differentially expressed genes (DEGs), of which 248 were upregulated and 190 were downregulated. DEGs were mainly involved in inflammatory related pathways, including neutrophil activation, neutrophil degranulation, neutrophil-mediated immunity, leukocyte chemotaxis, and lysosomes. The hub genes found through the method of degree in the PPI network showed that ITGB2 and ITGAM might play an important role in IPH. Receiver operating characteristic (ROC) results also showed a good performance of these two genes in the test and validation dataset. We found that the proportions of infiltrating immune cells in IPH and non-IPH samples differed, especially in terms of M0 and M2 macrophages. Immunohistochemistry and western blotting analysis showed that expression levels of ITGB2 and ITGAM increased significantly in carotid atherosclerotic plaques with IPH. CONCLUSION: ITGB2 and ITGAM are key hub genes of IPH and may play an important role in the biological process of IPH. Our findings advance our understanding of the underlying mechanisms of IPH pathogenesis and provide valuable information and directions for future research into novel targets for IPH diagnosis and immunotherapy.