Abstract
Purpose :
Elevated intraocular pressure (IOP) is a primary risk factor in the development of glaucoma. IOP is maintained by the trabecular meshwork (TM) through its ability to regulate resistance to aqueous humor outflow. The segmental nature of outflow in the TM has been well documented but investigation of molecular differences between the relative high flow (HF) and low flow (LF) regions have been limited by the small number of total cells in the TM. Here we establish a novel method to culture and characterize multiple paired HF and LF cell lines from human donor TM tissues.
Methods :
Anterior segments from human donor eyes were perfused in organ culture with amine-modified fluospheres to label HF and LF regions. HF regions from paired eyes were combined prior to culturing primary TM cells, as were LF regions. Eight paired sets of HF/LF cells were generated from eight biological donors ranging in age from 49-86. HF/LF cell lines were characterized by growth (cell count and MTT), proliferation, live/dead, myocilin expression in response to dexamethasone (dex) treatment and gene expression. Immunohistochemistry was used to determine protein expression differences in HF/LF cells.
Results :
Broadly, HF and LF cells have similar growth rates though it appears some LF lines grow slower than their HF counterparts. The cell lines generated from an 86 year old donor did not have the usual morphology of TM cells and grew slower than other lines. In particular, the LF cell line had a proliferation rate of just 4.9% while the HF line had a proliferation rate of 48%. Proliferation rates of cells with normal morphology range from 43-73%. Both HF and LF cell lines respond to dex treatment by increasing myocilin expression. Expression of 17 potential marker genes was determined by qRT-PCR on cells at early and late passages. Protein expression differences between HF/LF cells were found to corroborate the gene expression data.
Conclusions :
HF/LF cells have similar growth rates and both respond to dex treatment by increasing myocilin expression. HF cell lines may grow slightly faster than LF cells, though this is most dramatic in paired lines from older donors. Integrin alpha7, laminin alpha1, collagen VI, MMP2, MMP3 and MMP16 are potential markers for distinguishing HF cells from LF cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.