Figure 5. Validation of microarray analysis

A: Nine genes whose expression was upregulated (biglycan, VEGF, EMMPRIN, BMP-7, collagen VI alpha1, elastin, versican, and TGF-β2) or downregulated (thrombomodulin) in response to cyclical mechanical stretch were analyzed in independent experiments by real-time TaqMan PCR. The direction of expression changes in these genes was reproducible and consistent with microarray analysis. B: ELISA investigated if changes in mRNA synthesis reflected similar changes in protein production. VEGF and TGF-β2 were chosen as representative genes whose expression was altered by cyclical mechanical stretch. VEGF and TGF-β2 protein secretion was significantly increased in cell media supernatants of stretched lamina cribrosa cells compared to static controls (n=4). Mean control VEGF concentration was 241±66 pg/ml and mean stretch VEGF concentration was 407±148 pg/ml (n=4; p=0.03). Mean control TGF-β2 concentration was 87±19 pg/ml and mean stretch TGF-β2 concentration was 115±30 pg/ml (n=4; p=0.02). The asterisks indicate a statistically significant difference in protein secretion between stretched and control groups.