Figure 5

TGFβ and its receptors in ONH astrocytes. (a) Confirmation of three differentially expressed genes from the TGFβ-actin network (Figure 3a) by qRT-PCR in human ONH astrocytes: TGFBR2, SMAD3 and TGFBR1. Genes were normalized to 18S. Graphical representation of the relative mRNA levels in normal and glaucomatous AA and normal and glaucomatous CA astrocytes (n = 6, two-tailed t-test was used. Asterisk indicates p < 0.05). (b) Representative double immunofluorescent staining of TGFBR2 (red) and astrocyte marker GFAP (green) in sections of human ONH from an AA donor (51 year old male), AAG donor (70 year old male), CA donor (54 year old male) and CAG donor (76 year old male). Nuclei (blue) are stained with DAPI. Note granular staining of TGFBR2 in astrocytes (arrows) in the cribriform plates of the lamina cribrosa in AAG and CAG donors. Fewer astrocytes stain for TGFBR2 in the lamina cribrosa of CA donors. V, blood vessel; NB, nerve bundle. Scale bar 35 μm. (c) Representative western blots of astrocyte cell lysates with TGFBR2 antibody. β-Actin was used as a loading control. Note that AAG donors express more TGFBR2 than CAG donors. Normal AA and CA express lessTGFBR2 than glaucomatous donors. (d) Secreted TGFβ1 and TGFβ2 detected by ELISA. TGFβ2 is the primary form of TGFβ produced by ONH astrocytes. Secreted TGFβ1 is significantly higher in AA astrocytes compared to CA astrocytes (Asterisk indicates p < 0.05, two-tailed t-test); however, the increase in glaucomatous astrocytes compared to normal astrocytes is not significant. Secreted TGFβ2 levels are elevated significantly from normal AA astrocytes compared to all other donors (n = 24; asterisk indicates p < 0.05, two-tailed t-test).