Reactive changes in optic nerve astrocytes are mediated by CTGF, TGFβ2 and increasing substratum stiffness

Abstract

Purpose: Patients with primary open-angle glaucoma (POAG) show a stiffer peripapillary sclera, reactive astrocytes and a remodeled lamina cribrosa (LC). The changes are thought to be mediated by TGFβ2 and its downstream mediator CTGF. Recently we developed a murine glaucoma model by overexpressing CTGF in the anterior eye (βb1CTGF). In this study we investigated the glial lamina region of βb1CTGF mice, and the changes of astrocytes in response to CTGF and TGFβ2 as well as increasing substratum stiffness.

Methods: Tangential sections of the glial LC of 2-month-old βb1CTGF mice and their wild-type littermates (WT) were stained with phalloidin and antibodies against GFAP, CTGF and fibronectin (FN). Murine optic nerve (ON) astrocytes from CD1 mice were isolated, cultured and characterized by GFAP staining. The astrocytes were treated with TGFβ2 (1ng/ml) and CTGF (50ng/ml and 100ng/ml). In addition, the cells were seeded on PDMS substrata with different stiffness (10, 30 and 60 kPa). Treated cells were analyzed by Western blotting, real-time RT-PCR and immunohistochemistry. Wound healing assays were performed to analyze migration rate following growth factor treatment.

Results: βb1CTGF mice showed a massive increase in CTGF and GFAP in the glial LC when compared with WT mice. Moreover, an increase in FN staining and phalloidin-labeled actin was observed in the peripapillary sclera. Murine ON astrocytes reacted on increased substrate stiffness by increasing the synthesis of GFAP, vimentin and CTGF. Treatment of the cells with TGFβ2 and CTGF led to an enhanced migration rate. In addition, treatment resulted in an increased expression and synthesis of ECM proteins, including FN, tropoelastin, collagen type I and III. The in vitro findings correlated with those seen in the glaucoma mouse model.

Conclusions: We conclude that changes in the ECM of LC and peripapillary sclera alter their biomechanical properties and thereby induce reactive changes in resident astrocytes. The reactive changes induced by higher stiffness of their surrounding ECM give rise to a self-amplifying process that includes increased TGFβ2/CTGF signaling and leads to synthesis of ECM and cytoskeletal proteins, a process that in turn augments the stiffness at the optic nerve head (ONH). Such a scenario may finally result in a vicious circle as the causative mechanism for ONH deformation in POAG.