Abstract
Purpose :
In primary open-angle glaucoma optic nerve astrocytes show an increased reactivity after an increase of intraocular pressure (IOP), which leads to morphological changes and to alterations in the expression pattern of growth factors like CCN2/CTGF. The factors leading to optic nerve astrocyte reactivity are not completely understood. However, the mechanosensing capability of optic nerve astrocytes is important for their reactivity. Here we investigated whether Caveolin1 is involved in sensing changes in IOP and tissue stiffness in optic nerve astrocytes.
Methods :
We analyzed Caveolin-1 localization in the optic nerve and its expression pattern in the 2 month old βB1-CTGF1 glaucoma mouse model. Caveolin-1 expression was analyzed in murine optic nerve astrocytes cultured on substrates with increasing stiffness (8kPa, 50kPa) and after the treatment with the growth factor CCN2/CTGF (50 ng/ml) for 24h. The interplay of Caveolin-1 and CCN2/CTGF was observed by silencing experiments of both factors. In all experimental settings real-time RT-PCR, Western blot and immunohistochemistry were used. Statistical analysis was performed using two-tailed t-test. Data represented as mean ± SEM.
Results :
We observed that Caveolin-1 is predominantly localized in glial lamina. In the βB1-CTGF1 mouse model Caveolin-1 mRNA expression was significantly increased in the optic nerve head (wildtype: 1 ± 0.15, n=6; βB1-CTGF1: 6.65 ± 1.63, n=5; p=0.007), but not in the optic nerve (wildtype: 1 ± 0.29, n=15; βB1-CTGF1: 1.02 ± 0.30, n=16). In contrast, Caveolin-1 protein is significantly elevated in the entire optic nerve (wildtype: 1 ± 0.09; βB1-CTGF1: 2.53 ± 0.58; n=9 each; p=0.05). Cell culture analyses showed that murine optic nerve astrocytes respond to increasing substratum stiffness with an increase in Caveolin-1 expression (8kPa: 1; 50kPa: 1.93 ± 0.36; n=8 each; p=0.03). Treatment of murine optic nerve astrocytes with CCN2/CTGF resulted in an increased Caveolin-1 expression (control: 1; CCN2/CTGF: 1.61 ± 0.22; n=6 each; p=0.03) and protein synthesis (control 1; n=5 CCN2/CTGF: 1.8 ± 0.27, n=3; p=0.02), whereas silencing of CCN2/CTGF reduced the expression of Caveolin-1 (scrambled siRNA: 1; CCN2/CTGFsiRNA: 0.63 ± 0.10; n=5 each; p=0.002).
Conclusions :
We conclude that in optic nerve astrocytes Caveolin-1 and caveolae play a role in the mechanosensing of biomechanical alterations caused by increased IOP which can be modulated by growth factors.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.