Connexin43 Knockdown Accelerates Wound Healing but Inhibits Mesenchymal Transition after Corneal Endothelial Injury In Vivo

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, α-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 α-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 α-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 retrocorneal fibrous membrane formation.