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Carla Sanchez Martinez, Victoria E Tovell, Stephen J Tuft, Julie T Daniels; New insights into gene expression profiling of human aniridic corneal stromal cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1368.
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© ARVO (1962-2015); The Authors (2016-present)
Aniridia related keratopathy (ARK), caused by PAX6 haploinsufficiency, leads to corneal opacification and subsequently, sight loss. The biological mechanisms behind ARK progression are not yet fully understood. Previous research has not focused on the role of stromal cells on ARK. We hypothesise that aniridic corneal stromal cells may be affected and contribute to the development of ARK. For the first time, human aniridic corneal stromal cells (ACC) and normal corneal stromal cells (NCC) were cultured and studied inside a tissue equivalent (TE) to identify ARK specific gene and protein expression changes.
ACC (N=4) and NCC (N=4) were isolated and expanded from fresh and organ culture corneas, respectively. ACC were genotyped to identify their PAX6 variants. ACC and NCC were cultured inside a TE scaffold composed of compressed collagen type I for 21 days.Total RNA was extracted from ACC and NCC using TRIzol method and sent for RNA sequencing to GENEWIZ. The expression of relevant genes was confirmed by qPCR. ELISA was performed to assess MMP1 and MMP2 levels from the supernatant at day 7, 14 and 21. Two-tailed Student’s t-test (qPCR) and two-way repeated-measures ANOVA (ELISA) were used for statistical analysis.
ACC and NCC were successfully isolated, expanded, and cultured inside the TE. Previous functional studies have shown different cell organisation inside the TE; only 39% of the ACC population aligned to each other, compared to 75% of the NCC population. Two run-on mutations and two premature stops variants were identified in ACC. There were major differences between ACC and NCC transcriptomes (153 differentially expressed genes), including genes involved in angiogenesis, inflammatory response, regulation of apoptosis, and strikingly matrix disassembly and organization (p<0.005). The expression of MMP1 and MMP2, proteins with roles in stromal matrix remodeling, was downregulated in ACC compared to NCC (p<0.005). Accordingly, ACC secreted lower levels of MMP1 and MMP2 compared to NCC (p<0.05).
The ability of corneal stromal cells to align themselves and to remodel the extracellular matrix is essential to maintain the tissue homeostasis. Differences on MMPs expression and secretion profiles between the two cell populations suggest that their capacity to remodel the matrix may be impaired therefore contributing to ARK development.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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