O-GlcNAcylation-dependent liquid-liquid phase separation regulates the nuclear translocation of YAP to exacerbate vascular neointimal hyperplasia

Background: Suppressing the abnormal proliferation and migration of vascular smooth muscle cells (VSMCs), key pathological features of vascular neointimal hyperplasia (NIH), is an effective strategy for treating vascular insufficiency disorders caused by intimal remodeling. Increasing evidence suggests that Yes-associated protein (YAP) contributes to the abnormal proliferation and migration of VSMCs. However, the mechanisms by which YAP leads to NIH are poorly understood. Methods: An Immunofluorescence assay was used to detect the expression and distribution of YAP in mice or rats induced by ligation or balloon injury of the carotid artery. LC/MS, Co-immunoprecipitation (Co-IP), and confocal microscopy were used to evaluate O-GlcNAcylation, nucleation, and liquid-liquid phase separation (LLPS) of YAP, respectively. Protein-Protein Interaction Network (PPI) was used to predict potential binding substrates for YAP. The fluorescence recovery after photobleaching (FRAP) was applied to detect the binding of YAP to the substrate. Multiple biochemical analyses were conducted to unravel the underlying mechanisms. Results: YAP expression in synthetic-type VSMCs was highly increased in the injured artery. The up-regulated YAP in the nucleus of VSMCs increased transcription of the target gene CYR61. Knockdown of YAP and mutation of YAP O-GlcNAcylation sites in VSMCs in vitro attenuated PDGF-BB-induced abnormal proliferation and migration. This process was primarily due to the reduction of O-GlcNAcylation of YAP, which led to decreased LLPS of YAP and subsequently reduced the combination of YAP with the nuclear protein STAT3. Consequently, the nuclear translocation of YAP was affected, ultimately impacting the mRNA levels of CYR61, PCNA, OPN, and α-SMA. The small molecule hesperidin could inhibit YAP nuclear translocation and suppress NIH. Conclusion: Our findings revealed that O-GlcNAcylation-dependent LLPS regulates the nuclear translocation of YAP as a critical mechanism promoting NIH progression and may provide new strategies to prevent NIH.