Ophthalmology Research Laboratories, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
Brain Res Bull. 2010 Feb 15;81(2-3):262-72. doi: 10.1016/j.brainresbull.2009.10.007. Epub 2009 Oct 12.
Our previous data suggested the involvement of matrix metalloproteinase-10 (MMP-10) and cathepsin F (CTSF) in the basement membrane and integrin changes occurring in diabetic corneas. These markers were now examined in normal human organ-cultured corneas upon recombinant adenovirus (rAV)-driven transduction of MMP-10 and CTSF genes. Fifteen pairs of normal autopsy human corneas were used. One cornea of each pair was transduced with rAV expressing either CTSF or MMP-10 genes. 1-2 x 10(8) plaque forming units of rAV per cornea were added to cultures for 48 h with or without sildenafil citrate. The fellow cornea of each pair received control rAV with vector alone. After 6-10 days additional incubation without rAV, corneas were analyzed by Western blot or immunohistochemistry, or tested for healing of 5-mm circular epithelial wounds caused by topical application of n-heptanol. Sildenafil significantly increased epithelial transduction efficiency, apparently by stimulation of rAV endocytosis through caveolae. Corneas transduced with CTSF or MMP-10 genes or their combination had increased epithelial immunostaining of respective proteins compared to fellow control corneas. Staining for diabetic markers integrin alpha(3)beta(1), nidogen-1, nidogen-2, and laminin gamma2 chain became weaker and irregular upon proteinase transduction. Expression of phosphorylated Akt was decreased in proteinase-transduced corneas. Joint overexpression of both proteinases led to significantly slower corneal wound healing that became similar to that observed in diabetic corneas. The data suggest that MMP-10 and CTSF may be responsible for abnormal marker patterns and impaired wound healing in diabetic corneas. Inhibition of these proteinases in diabetic corneas may alleviate diabetic keratopathy symptoms.
我们之前的数据表明，基质金属蛋白酶-10（MMP-10）和组织蛋白酶 F（CTSF）参与了糖尿病角膜中基底膜和整合素的变化。现在，我们在正常的人器官培养角膜中，通过重组腺病毒（rAV）驱动 MMP-10 和 CTSF 基因的转导，检查了这些标志物。使用了 15 对正常尸检人眼角膜。每对角膜中的一只接受表达 CTSF 或 MMP-10 基因的 rAV 的转导。每只角膜加入 1-2×10(8)个 rAV 空斑形成单位，在有或没有西地那非枸橼酸盐的情况下培养 48 小时。每对角膜中的另一只接受单独载体的对照 rAV。在没有 rAV 的情况下再孵育 6-10 天后，通过 Western blot 或免疫组织化学分析角膜，或通过局部应用正庚醇引起的 5-mm 圆形上皮伤口愈合测试。西地那非显著增加了上皮转导效率，显然是通过刺激 rAV 通过小窝内吞作用。与对照角膜相比，转导 CTSF 或 MMP-10 基因或其组合的角膜具有增加的上皮免疫染色的相应蛋白。糖尿病标志物整合素 alpha(3)beta(1)、巢蛋白-1、巢蛋白-2 和层粘连蛋白 gamma2 链的染色在蛋白酶转导后变得更弱且不规则。蛋白酶转导的角膜中磷酸化 Akt 的表达减少。两种蛋白酶的联合过表达导致角膜伤口愈合明显减慢，类似于糖尿病角膜中的观察结果。数据表明，MMP-10 和 CTSF 可能是糖尿病角膜中异常标志物模式和伤口愈合受损的原因。在糖尿病角膜中抑制这些蛋白酶可能会减轻糖尿病角膜病变的症状。
