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Thania Bogarin, Sindhu Saraswathy, Ernesto Barron, Eric Barron, Jie J Zheng, Robert N Weinreb, Alex Huang; Human Scleral Fibroblast Steroid-Response and Cellular Migration Inhibition. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3189. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Steroid-response intraocular pressure (IOP) elevation can be seen after trabecular meshwork (TM) bypass or ablation suggesting distal targets. Previously, we showed that primary scleral fibroblasts responded similarly to TM cells after steroid treatment with diminished proliferation, increased cell-size, and altered filamentous actin stress fiber organization. Another known TM cellular steroid-response phenotype is diminished cellular migration so this was tested on scleral fibroblasts.
Primary scleral culture cells from 2 donors were grown under 3 treatment groups (n = 4 each): [A] dexamethasone, DEX [100 nM]; [B] media, MED; and [C] vehicle, DMSO for 7 days. After the 7th day, a linear scratch was made with WoundMaker, which was washed followed by continued drug treatment for 2.5 more days. Images were taken with IncuCyte Live-Cell Analysis System, and wound width, wound confluence, and relative wound density were determined by built-in software. Wound width was determined by taking the average distance between the edges of the initial scratch. Isolated cells, not contiguous to the native (unscratched) cell population on each side of the wound were excluded. Wound confluence percentage was the fractional wound area that was covered by cells. Relative wound density was measured as the ratio of cell-density in the wound to the cell density in the native (unscratched) region. Data = mean +/- SEM.
In wound width analysis, DEX-treated scleral fibroblast showed less healing and cell migration compared to MED- and DMSO- treated scleral fibroblasts (DEX= 1508 +/- 164; MED= 348 +/- 228; DMSO= 442 +/- 231 µm; DEX vs MED p <0.01, DEX vs DMSO p <0.01, MED vs DMSO p <0.78). DEX-treated cells also showed a decrease in wound confluence by 33-37% (p =0.005-0.01) and a decrease in relative wound density of 26-28% (p <0.5), compared to MED- and DMSO- treated cells. For all parameters, MED- and DMSO-treated control cells were not different (p = 0.7-0.8).
Primary scleral fibroblasts demonstrated steroid-response behaviors that negatively influenced cellular migration. This was very similar to original steroid-response behaviors reported for TM cells. These results continue to support our observations that scleral cells respond similarly to TM cells under steroid exposure, providing a possible explanation for continued steroid-induced IOP elevation even after TM bypass or ablation surgery.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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