Figure 3. Bone morphogenetic protein 7 (BMP7) treatment of retinal astrocyte cells increases markers of glial scar formation. A: Expression patterns for a panel of markers associated with reactive gliosis were compared in murine retinal astrocytes treated with sodium peroxynitrite or BMP7 for 24 or 36 h. For each experimental treatment, cells were treated with vehicle, sodium peroxynitrite, or BMP7 and real-time quantitative PCR (RT-qPCR) was undertaken. Levels of mRNA were normalized to internal control β 2 Microglobulin, and values were plotted relative to control levels. Each sample was run in triplicate and experiment repeated 3 times for each gene. Values represented are means ±SEM. Unpaired t test was performed between the control and treated groups with * denoting a p value<0.05 and ** denoting a p value<0.005. Any change above or below 1.0 indicates a change relative to control values. Peroxynitrite-treated cells showed statistically significant increases above 1.5-fold in mRNA levels of paired-homeobox 2 (Pax2), lipocalin2 (Lcn2), matrix metalloproteinase 9 (Mmp9), phosphacan (Pcan), S100β, toll like receptor 4 (Tlr4), tissue inhibitor of matix metalloproteinases 2 (Timp2), and glial fibrillary acidic protein (Gfap). Decreases in mRNA levels below 0.5-fold were noted for glutamate-aspartate transporter (Glast) in peroxynitrite-treated cells. In comparison, 24 h of BMP treatment led to a modest increase above the 1.5-fold level in neurocan (Ncan), Pcan, acid sensing ion channel 1 (Asic1), Glast, thioredoxin-interacting protein (Txnip), Pax2, and Gfap mRNA levels. By 36 h post BMP7-addition, Gfap levels had returned to baseline and Asic1, potassium inwardly rectifying channel 2.1 (Kir2.1), and Pax2 mRNA levels were reduced in comparison to vehicle-treated cells. Levels of Mmp11, Timp2, and Ncan were still increased in comparison to vehicle-treated cells. B: western blot analysis was performed for GFAP, TXNIP, and PAX2, with β-TUBULIN used as a loading control. Densitometric data shown are means+/−SEM of 3 trials. Unpaired t test was performed between the control and treated groups with * denoting a p value<0.05 and ** denoting a p value<0.005. Densitometric analysis of the blots showed a statistically significant increase in protein levels of GFAP in the peroxynitrite and 24 h BMP7 treatments. Significant increases in levels of PAX2 and TXNIP was observed in the peroxynitrite and 24 h BMP7 treatments, respectively. No statistically significant change was observed in the 36 h BMP7 treatment.