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Jr-Jiun Liou, Shenghuo Tien, Michael Yee, Paul R Kinchington, Christopher A Girkin, Jonathan Pieter Vande Geest; Manipulating Gene Expression of Human Lamina Cribrosa Cells and Astrocytes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6070. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Primary open angle glaucoma is a neurodegenerative disease that affects over 60 million people worldwide. It is more common in certain populations with prevalence rates of 2.4%, 3.6%, and 5.4% in those of European Descent, Hispanic Ethnicity, and African Descent, respectively. Previously, we have found that the microstructure and mechanical strain in lamina cribrosa (LC) is significantly different and that gremlin gene expression may be differentially regulated in these three populations. In this study, we aim to manipulate the gene expression of gremlin by delivering siRNA and shRNA via adeno-associated virus (AAV).
Human LC cells and astrocytes were isolated and cultured using an approved IACUC protocol. Immunostaining of gremlin was performed to examine protein expression in human LC cells and astrocytes. To evaluate the effect of transforming growth factor beta 2 (TGFβ-2) on human astrocytes, TGFβ-2 was added to the culture for 72 hours. To test AAV transfection efficiency, human LC cells and astrocytes were transfected with four different serotypes of AAV-CMV-GFP virus (AAV2, AAV5, AAV8, and AAV9) and the transfection efficiency was quantified by GFP signal.
Immunostaining results show that gremlin protein is present in both human LC cells and astrocytes (Figure 1A). Addition of TGFβ2 reduces gremlin protein expression in human astrocytes (Figure 1B). We found that both human LC cells and astrocytes are transfectable using an AAV virus, confirmed by GFP immunofluorescence (Figure 1C).
Prior work in the literature has shown that Gremlin is upregulated in the LC region of glaucomatous tissues. Our data suggest that TGFβ2 activity and extracellular matrix remodeling may contribute to the altered regulation of gremlin gene expression in astrocytes from donors of African Descent. We successfully delivered AAV into human LC cells and astrocytes and plan to examine the effect of gremlin knockdown on TGFβ2 activity and extracellular matrix remodeling in future work.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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