Connexin43 knockdown accelerates wound healing but inhibits mesenchymal transition after corneal endothelial injury in vivo
Connexin43 knockdown accelerates wound healing but inhibits mesenchymal transition after corneal endothelial injury in vivo
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DOI：
10.1167/iovs.07-0255
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发表时间：
2008-01-01
影响因子：
4.4
通讯作者：
Takamatsu, Tetsuro
中科院分区：
文献类型：
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作者：
Nakano, Yukiko;Oyamada, Masahito;Takamatsu, Tetsuro
目的.在大鼠角膜刮伤模型中，探讨连接蛋白43（Cx43）基因敲低对角膜内皮损伤的有效治疗作用。在角膜内皮中诱导刮伤，并进行免疫标记（ZO-1、α-SMA、Cx43）以分析伤口愈合期间Cx43表达的变化。将Cx43反义寡核苷酸（AS-ODN）、小干扰RNA（siRNA）或腺病毒（CMV-Cx43-mRFP 1）一次性注射到前房中，同时进行损伤，并通过免疫标记（ZO-1、Cx43）和碘化丙啶染色检查伤口闭合情况。用Ki 67免疫标记法检测角膜内皮细胞增殖情况。Cx43敲低治疗也进行无损伤，并检查其对Cx43表达和Ki 67免疫标记的影响。通过α-SMA免疫标记比较Cx43 AS-ODN和正义ODN处理的角膜中肌成纤维细胞的损伤后外观。在用Cx43 AS-ODN或siRNA处理的损伤后第3天，在六个角膜中的五个中观察到完全伤口闭合，而在对照角膜中在第3天没有观察到完全闭合（S-ODN，六个中的零个;或无义siRNA，六个中的零个）。一致地，使用腺病毒的Cx43过表达延迟伤口闭合。Cx43敲除增加了第1天Ki 67阳性增殖细胞的数量，而在第5天减少了α-SMA阳性肌成纤维细胞的数量。Cx43敲低无损伤降低Cx43表达并诱导体内内皮细胞增殖。这些结果表明，Cx43敲低诱导角膜内皮细胞增殖，但抑制损伤后的内皮-间充质转化/转化，表明Cx43敲低是一种新的治疗方法，用于加速伤口闭合和预防角膜后纤维膜形成。
PURPOSE. To explore connexin43 (Cx43) knockdown as an efficient treatment for corneal endothelial injury in an in vivo rat corneal scrape injury model.METHODS. Scrape injury was induced in the corneal endothelium, and immunolabeling (ZO-1, alpha-SMA, Cx43) was performed to analyze changes in Cx43 expression during wound healing. Single injection of Cx43 antisense oligodeoxynucleotide (AS-ODN), small interfering RNA (siRNA), or adenovirus (CMV-Cx43-mRFP1) was applied into the anterior chamber simultaneously with the injury, and wound closure was examined by immunolabeling (ZO-1, Cx43) and propidium iodide staining. Corneal endothelium proliferation on day 1 after injury was studied by Ki67-immunolabeling. Cx43-knockdown treatment was performed also without injury, and its effect on Cx43 expression and Ki67 immunolabeling was examined. The postinjury appearance of myofibroblasts in Cx43 AS-ODN- and sense-ODN-treated corneas was compared by alpha-SMA-immuno-labeling.RESULTS. Complete wound closures were observed in five of six corneas on day 3 after injury with either Cx43 AS-ODN or siRNA treatment, whereas no complete closure was observed on day 3 in the control corneas (S-ODN, zero of six; or nonsense siRNA, zero of six). Consistently, Cx43 overexpression using adenovirus delayed wound closure. Cx43 knockdown increased the number of Ki67-positive proliferating cells on day 1, whereas it decreased the number of alpha-SMA-positive myofibroblasts on day 5. Cx43 knockdown without injury decreased Cx43 expression and induced endothelial proliferation in vivo.CONCLUSIONS. These results show that Cx43 knockdown induces corneal endothelium proliferation but inhibits endothelial-mesenchymal transition/transformation after injury, suggesting that Cx43 knockdown is a new therapeutic approach for acceleration of wound closure and for prevention of retro-corneal fibrous membrane formation.