Saghizadeh Mehrnoosh, Kramerov Andrei A, Tajbakhsh Jian, Aoki Annette M, Wang Charles, Chai Ning-Ning, Ljubimova Julia Y, Sasaki Takako, Sosne Gabriel, Carlson Marc R J, Nelson Stanley F, Ljubimov Alexander V
Ophthalmology Research Laboratories, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
Invest Ophthalmol Vis Sci. 2005 Oct;46(10):3604-15. doi: 10.1167/iovs.04-1507.
To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I.
RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agilent Technologies, Palo Alto, CA). Gene expression in each diabetic corneal cRNA was assessed against pooled cRNA from 7 to 9 normal corneas. Select differentially expressed genes were validated by quantitative real-time RT-PCR (QPCR) and immunohistochemistry. Organ cultures were treated with a cathepsin inhibitor, cystatin C, or MMP-10.
More than 100 genes were upregulated and 2200 were downregulated in DR corneas. Expression of cathepsin F and hepatocyte growth factor (HGF) genes was increased in ex vivo and organ-cultured DR corneas compared with normal corneas. HGF receptor c-met, fibroblast growth factor (FGF)-3, its receptor FGFR3, tissue inhibitor of metalloproteinase (TIMP)-4, laminin alpha4 chain, and thymosin beta(4) genes were downregulated. The data were corroborated by QPCR and immunohistochemistry analyses; main changes of these components occurred in corneal epithelium. In organ-cultured DR corneas, cystatin C increased laminin-10 and integrin alpha(3)beta(1), whereas in normal corneas MMP-10 decreased laminin-10 and integrin alpha(3)beta(1) expression.
Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy.
除了先前描述的基质金属蛋白酶（MMP）-10、-3和胰岛素样生长因子（IGF）-I之外，鉴定在糖尿病视网膜病变（DR）供体的人角膜中异常表达的蛋白酶和生长因子。
从35个正常、糖尿病和DR尸检人角膜中离体或器官培养后分离RNA。使用22,000基因微阵列（安捷伦科技公司，加利福尼亚州帕洛阿尔托）分析扩增的cRNA。将每个糖尿病角膜cRNA中的基因表达与来自7至9个正常角膜的混合cRNA进行评估。通过定量实时RT-PCR（QPCR）和免疫组织化学验证选择的差异表达基因。器官培养物用组织蛋白酶抑制剂胱抑素C或MMP-10处理。
DR角膜中有100多个基因上调，2200个基因下调。与正常角膜相比，离体和器官培养的DR角膜中组织蛋白酶F和肝细胞生长因子（HGF）基因的表达增加。HGF受体c-met、成纤维细胞生长因子（FGF）-3、其受体FGFR3、金属蛋白酶组织抑制剂（TIMP）-4、层粘连蛋白α4链和胸腺素β4基因下调。这些数据通过QPCR和免疫组织化学分析得到证实；这些成分的主要变化发生在角膜上皮。在器官培养的DR角膜中，胱抑素C增加了层粘连蛋白-10和整合素α3β1，而在正常角膜中MMP-10降低了层粘连蛋白-10和整合素α3β1的表达。
组织蛋白酶F升高及其抑制剂在糖尿病角膜中产生更正常表型的能力表明这些角膜中蛋白水解增加。蛋白酶变化可能是由于DR角膜中生长因子如HGF和FGF-3的异常所致。蛋白酶和生长因子的特异性调节可减少糖尿病角膜上皮病变。
