TY - JOUR A1 - Dillinger, Andrea Elisabeth A1 - Mayer, Matthias A1 - Schneider, Magdalena A1 - Weber, Gregor R. A1 - Goeppner, Corinna A1 - Tamm, Ernst R. A1 - Shamonin (Chamonine), Mikhail A1 - Monkman, Gareth J. A1 - Fuchshofer, Rudolf T1 - Reactive changes in optic nerve astrocytes are mediated by CTGF, TGF beta 2 and increasing substratum stiffness JF - Investigative ophthalmology & visual science N2 - 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. Y1 - 2015 VL - 56 IS - 7 PB - ASSOC RESEARCH VISION OPHTHALMOLOGY INC ER - TY - JOUR A1 - Dillinger, Andrea Elisabeth A1 - Weber, Gregor R. A1 - Mayer, Matthias A1 - Schneider, Magdalena A1 - Göppner, Corinna A1 - Ohlmann, Andreas A1 - Shamonin (Chamonine), Mikhail A1 - Monkman, Gareth J. A1 - Fuchshofer, Rudolf A1 - Keller, Kate A1 - Lozano, Diana C. A1 - Clark, Abbot T1 - CCN2/CTGF-A Modulator of the Optic Nerve Head Astrocyte JF - Frontiers in cell and developmental biology (Front Cell Dev Biol.) N2 - In primary open-angle glaucoma (POAG), a neurodegenerative disease of the optic nerve (ON) and leading cause of blindness, the optic nerve head (ONH) undergoes marked structural extracellular matrix (ECM) changes, which contribute to its permanent deformation and to degeneration of ON axons. The remodeling process of the ECM causes changes in the biomechanical properties of the ONH and the peripapillary sclera, which is accompanied by an increased reactivity of the resident astrocytes. The molecular factors involved in the remodeling process belong to the Transforming growth factor (TGF)-β superfamily, especially TGF-β2. In previous publications we showed that TGF-β2 induced ECM alterations are mediated by Cellular Communication Network Factor (CCN)2/Connective Tissue Growth Factor (CTGF) and recently we showed that CCN2/CTGF is expressed by astrocytes of the ON under normal conditions. In this study we wanted to get a better understanding of the function of CCN2/CTGF under normal and pathologic conditions. To this end, we analyzed the glial lamina and peripapillary sclera of CCN2/CTGF overexpressing mice and studied the effect of CCN2/CTGF and increasing substratum stiffness on murine ON astrocytes in vitro. We observed enhanced astrocyte reactivity in the ONH, increased ECM protein synthesis in the peripapillary sclera and increased Ccn2/Ctgf expression in the ONH during the pathologic development in situ. CCN2/CTGF treatment of primary murine ON astrocytes induced a higher migration rate, and increase of ECM proteins including fibronectin, elastin and collagen type III. Furthermore, the astrocytes responded to stiffer substratum with increased glial fibrillary acidic protein, vimentin, actin and CCN2/CTGF synthesis. Finally, we observed the reinforced appearance of CCN2/CTGF in the lamina cribrosa of glaucomatous patients. We conclude that reactive changes in ONH astrocytes, induced by the altered biomechanical characteristics of the region, give rise to a self-amplifying process that includes increased TGF-β2/CCN2/CTGF signaling and leads to the synthesis of ECM molecules and cytoskeleton proteins, a process that in turn augments the stiffness at the ONH. Such a scenario may finally result in a vicious circle in the pathogenesis of POAG. The transgenic CTGF-overexpressing mouse model might be an optimal model to study the chronic pathological POAG changes in the ONH. KW - astrocytes KW - extracellar matrix KW - glaucoma KW - glial lamina KW - growth factors KW - optic nerve KW - stiffness Y1 - 2022 U6 - https://doi.org/10.3389/fcell.2022.864433 VL - 10 PB - frontiers ER -