Proteomic Analysis and 2-Hydroxyisobutyrylation Profiling in Metabolic Syndrome Induced Restenosis

Restenosis is the primary complication following stenting for coronary and peripheral arterial disease, posing an ongoing clinical challenge. Metabolic syndrome (MetS), characterized by metabolic disturbances, has been identified as an independent predictor for postoperative restenosis in coronary and carotid arteries, potentially due to endothelial dysfunction and augmented oxidative stress in cells, while its specific regulatory mechanism is still largely unknown. Lysine 2-hydroxyisobutyrylation (Khib), a recently identified posttranslational modification, plays a crucial role in transcriptional regulation and cellular metabolism. However, there is a lack of comprehensive analysis of the proteome and Khib modifications within restenotic vessels in the context of MetS, as well as in the understanding of the associated pathophysiology. In this study, we observed a significant upregulation of Khib in restenotic arteries induced by MetS, confirmed by animal and cellular experiments. Further, using high-throughput liquid chromatography-mass spectrometry, we catalogued 15,558 Khib sites across 2568 proteins, implicating a multitude of biological functions. Analysis revealed 2007 Khib sites on 1002 proteins with considerable differential modifications which are present within the cytoplasm and nucleus. Interestingly, proteins located in the mitochondria, endoplasmic reticulum, and cell membrane also exhibit distinct expression and modification profiles to varying extents that related to vascular smooth muscle contraction, platelet activation, and the PI3K-Akt signaling pathway. Notably, the level of COL1A1 protein detected in the protein-protein interaction pathway network and the level of Khib modification are diametrically opposed, suggesting a significant role in the disease's pathogenesis. This study provides the first comprehensive proteomic and Khib modification overview of MetS-related in-stent restenosis vasculature, offering key insights to inform novel therapeutic approaches for restenosis mitigation.