Weng Ping, Wen Yilin, Yuan Zhiyi, Ma Limei, Yang Liming, Li Chengju, Zhang Wanping, Yu Chao
College of Pharmacy, Chongqing Medical University, Chongqing, 400016, China.
Chongqing Key Laboratory for Pharmaceutical Metabolism Research, Chongqing,400016, China.
Theranostics. 2025 Jul 11;15(16):7956-7972. doi: 10.7150/thno.113303. eCollection 2025.
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. 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. 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 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. 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.
抑制血管平滑肌细胞（VSMCs）的异常增殖和迁移是治疗由内膜重塑引起的血管功能不全疾病的有效策略，而血管平滑肌细胞的异常增殖和迁移是血管新生内膜增生（NIH）的关键病理特征。越来越多的证据表明，Yes相关蛋白（YAP）促成了血管平滑肌细胞的异常增殖和迁移。然而，YAP导致NIH的机制尚不清楚。采用免疫荧光法检测颈动脉结扎或球囊损伤诱导的小鼠或大鼠中YAP的表达和分布。分别用液相色谱/质谱联用（LC/MS）、免疫共沉淀（Co-IP）和共聚焦显微镜评估YAP的O-连接N-乙酰葡糖胺化、成核和液-液相分离（LLPS）。利用蛋白质-蛋白质相互作用网络（PPI）预测YAP的潜在结合底物。应用光漂白后荧光恢复（FRAP）检测YAP与底物的结合。进行了多项生化分析以阐明潜在机制。在损伤动脉中，合成型血管平滑肌细胞中的YAP表达显著增加。血管平滑肌细胞核中上调的YAP增加了靶基因CYR61的转录。敲低血管平滑肌细胞中的YAP以及YAP O-连接N-乙酰葡糖胺化位点的突变减弱了血小板源性生长因子-BB（PDGF-BB）诱导的异常增殖和迁移。这一过程主要是由于YAP的O-连接N-乙酰葡糖胺化减少，导致YAP的液-液相分离减少，随后YAP与核蛋白信号转导和转录激活因子3（STAT3）的结合减少。因此，YAP的核转位受到影响，最终影响CYR61、增殖细胞核抗原（PCNA）、骨桥蛋白（OPN）和α-平滑肌肌动蛋白（α-SMA）的mRNA水平。小分子橙皮苷可抑制YAP核转位并抑制NIH。我们的研究结果表明，O-连接N-乙酰葡糖胺化依赖性液-液相分离调节YAP的核转位，这是促进NIH进展的关键机制，可能为预防NIH提供新策略。
