Department of Pathology, Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8.
Cardiovasc Pathol. 2013 Mar-Apr;22(2):156-66. doi: 10.1016/j.carpath.2012.06.008. Epub 2012 Aug 11.
Valve interstitial cells (VICs), the most prevalent cells in the heart valve, mediate normal valve function and repair in valve injury and disease. The Wnt3a/β-catenin pathway, important for proliferation and endothelial-to-mesenchymal transition in endocardial cushion formation in valve development, is up-regulated in adult valves with calcific aortic stenosis. Therefore, we tested the hypothesis that Wnt3a/β-catenin signaling regulates proliferation in adult VICs.
Porcine VICs were treated with 150 ng/ml of exogenous Wnt3a. To measure proliferation, cells were counted on day 4 posttreatment and stained for bromodeoxyuridine (BrdU) at 24 h posttreatment. β-Catenin small interfering RNA (siRNA) was used to knock down β-catenin expression. Apoptosis was measured with terminal deoxynucleotidyl transferase dUTP nick end labeling assay. To assess changes in β-catenin, cells were stained for β-catenin at days 1, 3, 6, and 9 posttreatment. Western blot for β-catenin was performed on whole cell, cytoplasmic, and nuclear extracts at day 4 posttreatment. To measure β-catenin-mediated transcription, TOPFLASH/FOPFLASH reporter assay was performed at 24 h posttreatment.
Wnt3a produced a significant increase in cell number at day 4 posttreatment and in the percentage of BrdU-positive nuclei at 24 h posttreatment. The increase in proliferation was abolished by β-catenin siRNA. Apoptosis was minimal in all conditions. Wnt3a produced progressively greater β-catenin staining as treatment length increased from 1 to 9 days. Wnt3a produced a significant increase in β-catenin protein in both whole cell and nuclear lysates after 4 days of treatment. Wnt3a significantly increased TOPFLASH/FOPFLASH reporter activity after 24 h of treatment.
Wnt3a/β-catenin signaling pathway is an important regulator of proliferation in adult VICs.
心脏瓣膜中最常见的细胞是瓣膜间质细胞（VIC），它在瓣膜损伤和疾病中介导正常的瓣膜功能和修复。Wnt3a/β-连环蛋白途径对于瓣膜发育中心内膜垫形成中的增殖和内皮细胞向间充质转化很重要，在成人钙化性主动脉狭窄的瓣膜中上调。因此，我们测试了 Wnt3a/β-连环蛋白信号通路调节成年 VIC 增殖的假设。
用 150ng/ml 的外源性 Wnt3a 处理猪 VIC。为了测量增殖，在处理后第 4 天计数细胞，并在处理后 24 小时用溴脱氧尿苷（BrdU）染色。用β-连环蛋白小干扰 RNA（siRNA）敲低β-连环蛋白表达。用末端脱氧核苷酸转移酶 dUTP 缺口末端标记法测量细胞凋亡。为了评估β-连环蛋白的变化，用β-连环蛋白在第 1、3、6 和 9 天进行细胞染色。在处理后第 4 天进行全细胞、细胞质和核提取物的β-连环蛋白的 Western blot。在处理后 24 小时进行 TOPFLASH/FOPFLASH 报告基因检测，以测量β-连环蛋白介导的转录。
Wnt3a 在处理后第 4 天显著增加细胞数量，并在处理后 24 小时增加 BrdU 阳性核的比例。β-连环蛋白 siRNA 可消除增殖的增加。在所有条件下，细胞凋亡都很少。Wnt3a 在处理时间从 1 天延长至 9 天时，β-连环蛋白染色逐渐增加。Wnt3a 在处理 4 天后，在全细胞和核提取物中均显著增加β-连环蛋白蛋白。Wnt3a 在处理后 24 小时显著增加 TOPFLASH/FOPFLASH 报告基因活性。
Wnt3a/β-连环蛋白信号通路是调节成年 VIC 增殖的重要调节剂。
