Kirwan Ruaidhrí P, Wordinger Robert J, Clark Abbot F, O'Brien Colm J
Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland.
Mol Vis. 2009;15:76-88. Epub 2009 Jan 15.
Marked extracellular matrix (ECM) remodeling occurs in the human optic nerve head in primary open angle glaucoma (POAG). The glial fibrillary acid protein (GFAP) negative lamina cribrosa cell may play an important role in this remodeling process. We report the first study of global and ECM-focused gene transcription differentials between GFAP-negative lamina cribrosa (LC) cells from normal and POAG human donors.
GFAP-negative LC cell lines were generated from the optic nerve tissue of four normal (n=4) and four POAG (n=4) human donors. Using Affymetrix U133A arrays the transcriptional profile between the normal and diseased groups were compared. Bioinformatic analysis was performed using robust multichip average (RMA Express) and EASE/David. Real time TaqMan PCR and immunohistochemistry analyses were performed to validate the microarray data.
183 genes were upregulated by greater than 1.5 fold and 220 were down regulated by greater than 1.5 fold in the POAG LC cells versus normal controls. Upregulated genes in POAG LC cells included, transforming growth factor beta 1 (TGFbeta1), secreted acid protein cysteine rich (SPARC), periostin (POSTN), thrombospondin-1 (THBS1), cartilage linking protein-1 (CRTL-1), and collagen type I (COL1A1), collagen type V (COL5A1), and collagen type XI (COL11A1). Downregulated ECM genes in POAG included fibulin 1 (FBLN1), decorin (DCN), and collagen type XVIII (COL18A1). All TaqMan PCR validation assays were significant (*p<0.05) and consistent with the array data. Immunohistochemistry of one target (periostin) confirmed its differential expression at the protein level in POAG optic nerve head tissue compared with non-glaucomatous controls. Functional annotation and over-representation analysis identified ECM genes as a statistically over-represented class of genes in POAG LC cells compared with normal LC cells.
This study reports for the first time that POAG LC cells in-vitro demonstrate upregulated ECM and pro-fibrotic gene expression compared with normal LC cells. This may be a pathological characteristic of this cell type in POAG in-vivo. We believe that the LC cell may be a pivotal regulator of optic nerve head ECM remodeling in POAG and an attractive target for molecular therapeutic strategies in the future.
在原发性开角型青光眼（POAG）患者的人视神经乳头中，发生了显著的细胞外基质（ECM）重塑。神经胶质纤维酸性蛋白（GFAP）阴性的筛板细胞可能在这一重塑过程中发挥重要作用。我们首次报道了对来自正常和POAG人类供体的GFAP阴性筛板（LC）细胞之间的整体和聚焦于ECM的基因转录差异的研究。
从四名正常（n = 4）和四名POAG（n = 4）人类供体的视神经组织中生成GFAP阴性LC细胞系。使用Affymetrix U133A阵列比较正常组和疾病组之间的转录谱。使用稳健多芯片平均法（RMA Express）和EASE/David进行生物信息学分析。进行实时TaqMan PCR和免疫组织化学分析以验证微阵列数据。
与正常对照相比，POAG LC细胞中183个基因上调超过1.5倍，220个基因下调超过1.5倍。POAG LC细胞中上调的基因包括转化生长因子β1（TGFβ1）、富含半胱氨酸的分泌酸性蛋白（SPARC）、骨膜蛋白（POSTN）、血小板反应蛋白-1（THBS1）、软骨连接蛋白-1（CRTL-1），以及I型胶原（COL1A1）、V型胶原（COL5A1）和XI型胶原（COL11A1）。POAG中下调的ECM基因包括纤连蛋白1（FBLN1）、核心蛋白聚糖（DCN）和XVIII型胶原（COL18A1）。所有TaqMan PCR验证试验均具有显著性（*p<0.05），且与阵列数据一致。对一个靶点（骨膜蛋白）的免疫组织化学证实了其在POAG视神经乳头组织中与非青光眼对照相比在蛋白质水平上的差异表达。功能注释和过度表达分析确定，与正常LC细胞相比，ECM基因在POAG LC细胞中是统计学上过度表达的一类基因。
本研究首次报道，与正常LC细胞相比，体外培养的POAG LC细胞表现出ECM和促纤维化基因表达上调。这可能是POAG体内这种细胞类型的病理特征。我们认为，LC细胞可能是POAG视神经乳头ECM重塑的关键调节因子，也是未来分子治疗策略的一个有吸引力的靶点。
