Kenney M Cristina, Chwa Marilyn, Atilano Shari R, Tran Annie, Carballo Marilee, Saghizadeh Mehrnoosh, Vasiliou Vasilis, Adachi Wakako, Brown Donald J
Department of Ophthalmology, University of California Irvine, Medical Center, 101 The City Drive, Orange, CA 92868, USA.
Invest Ophthalmol Vis Sci. 2005 Mar;46(3):823-32. doi: 10.1167/iovs.04-0549.
The mRNA levels of antioxidant enzymes, matrix metalloproteinases, cathepsin V/L2, and tissue inhibitor of matrix metalloproteinases (TIMPs) were determined in keratoconus and normal corneas. Protein levels or enzyme activities were analyzed when RNA levels were different.
A total of 25 physiologic (normal) and 32 keratoconus corneas were studied. mRNAs were analyzed by semiquantitative reverse transcription-polymerase chain reaction and Southern blot analysis. Proteins were assessed by immunohistochemistry and/or Western blot analysis. Catalase activity was measured in corneal extracts. Antioxidant enzymes examined were catalase, superoxide dismutase (SOD)-1, SOD3, glutathione reductase, glutathione S-transferase and aldehyde dehydrogenase 3A1. Degradative enzymes examined were cathepsin V/L2 and matrix metalloproteinase (MMP)-1, -2, -7, -9, and -14. Tissue inhibitor of matrix metalloproteinase (TIMP)-1, -2, and -3 were also examined.
Keratoconus corneas exhibited a 2.2-fold increase of catalase mRNA level (P < 0.01) and 1.8-fold of enzyme activity (P < 0.03); a 1.5-fold increase of cathepsin V/L2 mRNA (P < 0.03) and abnormal protein distribution; and a 1.8-fold decrease of TIMP-1 mRNA (P < 0.05) and 2.8-fold decrease of protein (P < 0.0001) compared with normal (physiologic) corneas. RNA levels for other antioxidant and degradative enzymes were similar between normal and keratoconus corneas.
Keratoconus corneas have elevated levels of cathepsins V/L2, -B, and -G, which can stimulate hydrogen peroxide production, which, in turn, can upregulate catalase, an antioxidant enzyme. In addition, decreased TIMP-1 and increased cathepsin V/L2 levels may play a role in the matrix degradation that is a hallmark of keratoconus corneas. The findings support the hypothesis that keratoconus corneas undergo oxidative stress and tissue degradation.
测定圆锥角膜和正常角膜中抗氧化酶、基质金属蛋白酶、组织蛋白酶V/L2以及基质金属蛋白酶组织抑制剂（TIMPs）的mRNA水平。当RNA水平存在差异时，分析蛋白质水平或酶活性。
共研究了25个生理状态（正常）角膜和32个圆锥角膜。通过半定量逆转录-聚合酶链反应和Southern印迹分析来检测mRNA。通过免疫组织化学和/或蛋白质印迹分析来评估蛋白质。在角膜提取物中测量过氧化氢酶活性。检测的抗氧化酶包括过氧化氢酶、超氧化物歧化酶（SOD）-1、SOD3、谷胱甘肽还原酶、谷胱甘肽S-转移酶和醛脱氢酶3A1。检测的降解酶包括组织蛋白酶V/L2和基质金属蛋白酶（MMP）-1、-2、-7、-9和-14。还检测了基质金属蛋白酶组织抑制剂（TIMP）-1、-2和-3。
与正常（生理状态）角膜相比，圆锥角膜的过氧化氢酶mRNA水平增加了2.2倍（P < 0.01），酶活性增加了1.8倍（P < 0.03）；组织蛋白酶V/L2 mRNA增加了1.5倍（P < 0.03）且蛋白质分布异常；TIMP-1 mRNA减少了1.8倍（P < 0.05），蛋白质减少了2.8倍（P < 0.0001）。正常角膜和圆锥角膜中其他抗氧化酶和降解酶的RNA水平相似。
圆锥角膜中组织蛋白酶V/L2、-B和-G水平升高，可刺激过氧化氢生成，进而上调抗氧化酶过氧化氢酶。此外,TIMP-1水平降低和组织蛋白酶V/L2水平升高可能在圆锥角膜标志性的基质降解中起作用。这些发现支持圆锥角膜经历氧化应激和组织降解这一假说。
