Lv Yu, Wang Xia, Zeng Youjie, Tang Zizhao, Nie Fangqin, Guo Ren
Department of Pharmacy, The Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, China; Department of Orthopaedics, Shanghai Bone Tumor Institute, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
Department of Pharmacy, The Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, China.
Tissue Cell. 2025 Apr;93:102682. doi: 10.1016/j.tice.2024.102682. Epub 2024 Dec 12.
The phenotypic transition of vascular smooth muscle cells (VSMCs) from a quiescent, contractile type to a secretory phenotype with high proliferation and mobility is a key event in vascular remodeling. PF-477736 is an ATP-competitive inhibitor of Chk1 which induces the accumulation of DNA damage by increasing the level of replicative stress, and ultimately inhibiting cell proliferation or causing cell death. Although this compound has been utilized as an anti-tumor drug, its role in vascular remodeling remains unclear.
In vitro, Human aortic smooth muscle cell line (HAVSMC) and primary rat aortic smooth muscle cells were used to establish phenotype transformation model with PDGF-bb; Western blot was used to detect the expression of VSMCs phenotype marker α-SMA, Vimentin; MTT and EdU assays were used to evaluate the proliferation ability of VSMCs; wound healing assay was used to evaluate the migration ability of VSMCs. In vivo, we established ballon injury of carotid artery in rats, and the function of the PF-477736 was evaluated by several histological stainings.
The results exhibit that PF-477736 effectively inhibited VSMCs phenotypic transition, resulting in G1/S phase arrest and decreased proliferation and migration ability of VSMCs. Furthermore, while PDGF-bb down-regulated p53 protein and up-regulated CD44 expression, PF-477736 significantly countered these effects. Pretreatment of VSMCs with p53 siRNA blocked the effect of PF-477736, up-regulated the expression of CD44, and promoted VSMCs' proliferation and migration. Conversely, CD44 silencing through siRNA mitigated the phenotypic transition of VSMCs. In addition, the H&E, Masson' staining and the immunohistochemistry of PCNA, p53 and CD44 showed that PF-477736 substantially inhibits vascular remodeling in the balloon injury model.
Our findings show that PF-477736 exerts anti-vascular remodeling effect by inhibiting phenotypic transition through the Chk1/p53/CD44 pathway in VSMCs, providing novel therapeutic strategies for preventing and treating vascular remodeling.
血管平滑肌细胞（VSMCs）从静止的收缩型向具有高增殖和迁移能力的分泌表型的转变是血管重塑的关键事件。PF-477736是Chk1的一种ATP竞争性抑制剂，它通过增加复制应激水平诱导DNA损伤积累，最终抑制细胞增殖或导致细胞死亡。尽管该化合物已被用作抗肿瘤药物，但其在血管重塑中的作用仍不清楚。
在体外，用人主动脉平滑肌细胞系（HAVSMC）和原代大鼠主动脉平滑肌细胞建立PDGF-bb诱导的表型转化模型；采用蛋白质免疫印迹法检测VSMCs表型标志物α-SMA、波形蛋白的表达；采用MTT法和EdU法评估VSMCs的增殖能力；采用划痕实验评估VSMCs的迁移能力。在体内，我们建立大鼠颈动脉球囊损伤模型，并通过多种组织学染色评估PF-477736的作用。
结果表明，PF-477736有效抑制VSMCs表型转化，导致G1/S期阻滞，降低VSMCs的增殖和迁移能力。此外，虽然PDGF-bb下调p53蛋白并上调CD44表达，但PF-477736显著对抗这些作用。用p53 siRNA预处理VSMCs可阻断PF-477736的作用，上调CD44表达，并促进VSMCs的增殖和迁移。相反，通过siRNA沉默CD44可减轻VSMCs的表型转化。此外，苏木精-伊红染色、Masson染色以及PCNA、p53和CD44的免疫组化结果显示，PF-477736在球囊损伤模型中显著抑制血管重塑。
我们的研究结果表明，PF-477736通过抑制VSMCs中Chk1/p53/CD44途径的表型转化发挥抗血管重塑作用，为预防和治疗血管重塑提供了新的治疗策略。
