Abstract Body (Do not enter title and authors here): Background: After percutaneous coronary intervention (PCI), 5-10% of patients experience in-stent restenosis (ISR), and among these, 10-20% develop recurrent in-stent restenosis (RISR). Proteins could be encapsulated into exosomes and participate in intercellular signaling, affecting various progression of cardiovascular diseases. However, the effect of exosome-derived proteins on RISR progression needs further exploration.
Aims: Vascular endothelial injury initiates restenosis post-PCI, with the excessive proliferation and migration of vascular smooth muscle cells (VSMCs) playing a central role. To elucidate the mechanisms by which exosomes derived from endothelial cells influence the proliferation and migration of VSMCs, thereby contributing to the exacerbation of RISR.
Methods: Firstly, coronary plasma was collected from 8 RISR patients and 8 healthy controls. Exosomes were isolated using ultracentrifugation techniques. Protein components in exosomes were sequenced by four-dimensional data-independent acquisition (4D-DIA) quantitative proteomics. Exosomes with high TGF-β1 were co-cultured with primary VSMCs to assess their functional impact on cell behavior. After 24 hours of serum starvation to synchronize cell cycles, the cells were treated with PKH67-labeled exosomes. Subsequent confocal microscopy analysis to evalulate the presence of PKH67-labeled exosomes in VSMCs. Next, we performed cell proliferation assays (CCK-8) and migration assays (wound healing and transwell migration) to evaluate the effects of exosomal transfer on VSMC activity. The expression of TGF-β1, MYH9 and Smad2/3 pathway with their phosphorylation status in exosome-treated VSMCs were tested using WB assays.
Results:
Exosomes from RISR patients, with high TGF-β1 expression, were originated from endothelial cells and could be internalized by VSMCs. TGF-β1 activated Smad2/3 pathway and then promoted MYH9 expression. In addition, overexpression of TGF-β1 boosted the expression of MYH9, while the knockdown of TGF-β1 has the opposite effect. The functional rescue experiment validated that TGF-β1 can regulate downstream MYH9, and participates in the proliferation and migration of VSMCs via Smad2/3 pathway.
Conclusion: Our study suggests that exosome-derived, endothelial cell-originated TGF-β1 is essential to VSMCs proliferation and migration. This process is mediated via TGF-β1- Smad2/3-MYH9 axis.