Abstract
Purpose: :
The expression of Serum Amyloid A (SAA) is increased in glaucomatous TM and aqueous humor. Increased SAA caused elevated intraocular pressure in ex vivo perfusion cultured human donor eyes and in vivo in a viral overexpressed mouse eyes, providing evidence for linkage of overexpression of SAA to glaucomatous ocular hypertension. In this study we investigated the effects of SAA on genome-wide gene expression in TM cells to determine the signaling pathway(s) that are involved in SAA-induced ocular hypertension.
Methods: :
Two human TM cell lines cultured in 75 cm2 flasks were treated with rhSAA (40ug/ml) in triplicate for 24 hr. RNA was extracted from the cells and 15 µg of each RNA was used for gene expression analysis using Affymetrix Genechips (HG-U133A & B) in triplicate (3 normal vs. 3 treated samples). Microarray data were analyzed by MAS5.0, SAM (Significance Analysis of Microarrays), GeneSpring and Biobase programs. The microarray results were confirmed by real time QPCR for selected genes using the same RNA that was used for microarray, as well as RNA from 5 additional TM cell lines.
Results: :
Reproducibility among the replicate (3 control and 3 treatment) microarray chips was excellent. The correlation coefficient (r2) between any two control or treated chips was high (0.97 to 0.99). SAM analysis revealed 200 significantly changed genes (Delta =7.4, fold change ≥2, false discovery rate ~ 1%) in SAA treated TM cells compared to controls, with 169 genes upregulated and 31 genes downregulated. The most significantly affected gene classes were physiological process (161 genes), cellular process (151 genes), response to stimulus (68 genes), and regulation of biological process (53 genes). They include genes that are primarily involved in the caspase network (106 genes), TLR3 pathway (38 genes), Apo2L pathway (34 genes), and cholesterol biosynthesis/metabolism (38 genes). SAA induced matrix metalloproteinases (MMPs), cytokines, cytokine receptors, and nuclear factor B (NF- B). Among the genes increased by SAA treatment were IL-6 (30.6 fold, p<0.0001), IL-8 (22.8 fold, p<0.0001), TNFAIP3 (9.5 fold, p<0.0001), NFKB2 (2.1 fold, p<0.0002) and MMP3 (4.7 fold, p<0.0003). Differential expression of several of these genes was confirmed by QPCR. Differential expression of IL-8, TNFAIP3, and TIMP3 was confirmed by QPCR in five additional TM cell lines treated with SAA.
Conclusions: :
We have shown that SAA significantly impacts genome-wide gene expression in TM cells. Identification of SAA regulated genes and signaling pathways may provide important insights into SAA-induced ocular hypertension and glaucoma pathogenesis.
Keywords: gene/expression • trabecular meshwork • gene microarray