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Amanda Mae Hurley, Monte J Radeke, Pete Coffey; Growth differentiation factor 6 (GDF6) is a novel inducer of the epithelial-to-mesenchymal transition in retinal pigmented epithelial cells.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6047.
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As retinal pigmented epithelium (RPE) cells are passaged, they undergo an irreversible epithelial-to-mesenchymal transition (EMT). We have shown previously, that GDF6, a member of the transforming growth factor-beta (TGFβ) family, is highly upregulated in RPE cells that have undergone EMT. We hypothesize that GDF6 plays an integral role in the irreversible transition of an RPE cell from an epithelial state to a mesenchymal state.
Passage 0 fetal RPE cells were transduced with a lentiviral construct encoding GDF6 or an empty vector control. The GDF6 positive cells were collected, re-plated at a high density, and allowed to differentiate for 32 days. At this point images were obtained for pigmentation analysis and cells were harvested for RNA. Pigmentation was analyzed using Matlab software. The images were binarized and the number of back pixels was divided by the total number of pixels. qRT-PCR was performed using a variety of EMT and RPE markers. Student’s T tests were used to determine statistical significance
RPE transduced with GDF6 produce a significantly lower level of pigmentation (21%) than cells infected with an empty vector control (68%, p=0.0001). This reduction in pigmentation is accompanied with a change in cell morphology; where the control cells maintain a cuboidal morphology and the GDF6 secreting cells have a spindle-like morphology. qRT-PCR analysis reveals that RPE transduced with GDF6 significantly upregulate known EMT markers like ACTA2, CTGF, and COL1A1 and downregulate classical RPE markers such as LRAT, APOE, and PMEL compared to control cells.
GDF6 is a novel inducer of EMT in RPE. Cells that overexpress GDF6 will prematurely undergo EMT, simultaneously downregulating traditional RPE markers while upregulating EMT and wound response markers. Inhibition of GDF6 may be integral in prolonging the integrity of RPE cultures.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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