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Declan Ilan Hesson, Yang Liu, Abbot F Clark; Canonical Wnt signaling in optic nerve head astrocytes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5671. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Canonical Wnt signaling has been explored in areas of the eye like the trabecular meshwork and ciliary body during glaucomatous pathology, but it has not been explored in the optic nerve head (ONH). Complement protein 1, subunit q (C1q) has been shown to be an activator of the canonical Wnt pathway leading to an increase in fibrosis in various tissues. As C1q concentration increases in blood serum and central nervous system with an increase in age, and is increased in the glaucomatous ONH, C1q activation of canonical Wnt signaling may be an important factor in glaucomatous pathology and may be a source of increased fibrosis in the ONH. Therefore, we hypothesize that a functional canonical Wnt signaling pathway is expressed in the ONH, and C1q is an activator of this pathway which increases fibrosis of the ONH.
Primary human ONH astrocytes (ONHA) from human donor eyes were cultured and characterized. When confluent, cells were serum starved overnight and then treated for 48 hours with 100 nM Wnt3a or left untreated as a control. Following treatment, cells were collected, and cytosolic and nuclear fractions separated. Fractions were then western blotted and probed for β-Catenin. Bands were analyzed via densotometry and fold changes in expression were compared to control cells. Additional primary human ONHA were cultured and, when confluent, serum starved overnight. Cells were treated with 100 nM C1q, with or without the Wnt antagonist DKK1 (100 nM), and the expression of fibronectin, laminin, and collagens I and IV was determined via western blotting and immunohistochemistry.
In a single primary human ONHA strain, β-Catenin expression increased 1.3-fold when treated with Wnt3a and 1.42-fold when treated with C1q in the cytoplasmic fraction compared to control. β-Catenin expression increased 1.3-fold when treated with Wnt3a and 1.39-fold when treated with C1q in the nuclear fraction compared to control. Immunohistochemistry staining for fibronectin increased following treatment with C1q compared to the control. Additional ONHA strains will be treated to determine statistical significance.
Our preliminary results support our hypothesis that a functional canonical Wnt signaling pathway is expressed in the ONH. Additional cell strains will need to be examined to fully determine presence of a functional canonical Wnt signaling pathway as well as determining if C1q activates the pathway in these ONH cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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