8.2.1　体内ATO促进VSMCs从合成型向收缩型转化

不同种类血管支架（BMS、PMS、AES）植入新西兰大白兔颈动脉1个月和3个月后，对支架处血管的平滑肌表型因子（α-SMA）进行HE染色及免疫荧光染色（绿色），如图8-2所示。通过荧光强度评估其表达量；统计分析新生内膜处VSMCs的纵横比以表征其细胞的形状。结果发现ATO药物洗脱支架可以明显促进α-SMA的表达，并使VSMCs较早地完成了向成熟VSMCs的纺锤形表型改变。

图8-2　AES在体内抑制支架内再狭窄及诱导新内膜处平滑肌细胞的表型分化。（a）支架植入血管1个月时的HE染色。（b）支架植入血管1个月时内膜增生统计图（n=3）。（c）支架植入1个月时行α-SMA的免疫荧光染色（绿色）。（d）荧光强度统计图（n=6）。（e）支架植入3个月时行α-SMA的免疫荧光染色（绿色）。（f）细胞纵横比统计图（n=30）。“L”代表血管腔；BMS：裸金属支架；PMS：聚合物涂层金属支架；AES：ATO药物洗脱支架；标尺长度分别为200 μm（a）、100 μm（c）和50 μm（e）；“ns”表示无显著性差异，p值＜0.01（**），0.001（***），＜0.000 1（****）。［引　自：Zhao Y, et al. A novel mechanism of inhibiting instent restenosis with arsenic trioxide drug-eluting stent: enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway［J］. Bioact Mater, 2021, 6（2）: 375-385.］
Figure 8-2　In vivo, AES inhibits in-stent restenosis and induces differentiation of smooth muscle cells in neointima.（a）Hematoxylin and eosin（HE）staining of vascular after stent implantation for 1 month, scar bar = 200 μm.（b）The statistical results of neointima/media area ratio for HE staining after stent implantation for 1 month, n=3.（c）Immunofluorescent staining with α-SMA（green）after stent implantation for 1 mo, scar bar = 100 μm.（d）The statistical results of mean fluorescence intensity of α-SMA after stent implantation for 1 mo in neointima, n=6.（e）Immunofluorescent staining with α-SMA（green）after stent implantation for and 3 months, scar bar = 50 μm.（f）The statistical results of cell aspect ratio（cell long/short axis ratio）of VSMCs after stent implantation for 3 months in neointima, n=30. “L” for the lumen. BMS（bare metal stent）, PMS（polymer coating-metal stent）, and AES（arsenic trioxide-drug eluting stent）; “ns” means no significance, and p values＜0.01（**）,＜0.001（***）and＜0.000 1（****）. ［Adapted from: Zhao Y, et al. A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway［J］. Bioact Mater, 2021, 6（2）: 375-385.］(https://www.daowen.com)

图8-3　在体外，三氧化二砷诱导表型调节，并抑制合成型VSMC的细胞活力。（a）为A7r5血管平滑肌细胞行α-SMA免疫荧光染色图。（b）为图（a）中免疫荧光强度统计图。（c）为ATO处理A7r5后细胞的纵横比统计图。（d）和（e）分别为蛋白免疫印迹及蛋白表达量统计图。（f）为猪原代冠状动脉平滑肌细胞（PCASMCs）的提取及鉴定。（g）为PCASMCs在有无PDGF刺激下细胞的增殖特性。（h）和（i）分别为收缩型PCASMCs及合成型PCASMCs的诱导示意图及细胞活力测定。其中，A0、A2、A4和A6代表ATO的浓度分别为 0、2、4和6 μmol/L。“ns”表示无显著性差异，P值＜0.01（**），0.001（***），＜0.000 1（****）。［引自：Zhao Y, et al. A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway［J］. Bioact Mater, 2021, 6（2）: 375-385.］
Figure 8-3　In vitro, arsenic trioxide induces phenotype modulation, and inhibits cell viability of synthetic phenotype VSMCs.（a）α-SMA immunostaining of A7r5 with or without ATO treating for 1 d.（b）The statistical results of mean fluorescence intensity of α-SMA in（a）.（c）The statistical results of cell aspect ratio（cell long/short axis ratio）of A7r5 with or without ATO treating for 1 d.（d）and（e）, Protein levels of SM22α and α-SMA were determined by WB after ATO treating for 1 d.（f）The extraction and identification of primary porcine coronary artery smooth muscle cells（PCASMCs）, scar bar =100 μm.（g）The proliferation profile of PCASMCs, scar bar =100 μm.（h）The schematic diagram for the phenotype induction of PCASMCs.（i）The viability of contractile- and synthetic- phenotype PCASMCs on different concentration of ATO for 1 and 3 d. A0, A2, A4 and A6 represent 0, 2, 4 and 6 μmol/L of ATO, respectively. “ns” means no significance, p values＜0.01（**）,＜0.001（***）and＜0.000 1（****）. ［Adapted from: Zhao Y, et al. A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway［J］. Bioact Mater, 2021, 6（2）: 375-385.］