Abstract
Purpose :
A recent study of aqueous humor (AH) composition in primary open angle glaucoma identified uteroglobin (UG) as a protein of interest. Whilst little is known about UG function, the literature indicates a possible role in control of trabecular meshwork (TM) contraction, thus affecting AH outflow. We hypothesised that treating TM cells in a 3D collagen gel with uteroglobin would alter their contractibility.
Methods :
Commercial human TM cells (hTMCs) were characterised by analysis of TM marker gene expression (AQP1, MGP, TIMP2, MMP3, LMX1B and CHI3L1), increased myocilin expression in response to 3-day dexamethasone exposure (Dex; 100 nM) and 3D collagen gel contraction. Expression of UG receptors FPR2, LIMBR1L and PTGDR was assessed using quantitative PCR (qPCR) and immunofluorescence (IF). HTMCs treated with 1 or 2 ug/ml UG were embedded in 3D collagen gels and contraction was photographed at 6, 12, 24, 36, 48, 72 and 96 hours. Both subconfluent and 7-day contact-inhibited hTMCs were tested. Treatment with ROCK inhibitor Y-27632 (100 uM) was used to inhibit contraction, and HeLa and human dermal fibroblasts (HDFs) were used as negative and positive control cell lines, respectively. All experiments were conducted in triplicate and repeated three times (n=3). Collagen contraction was quantified using ImageJ and significance was assessed using a two-way ANOVA in Prism6.
Results :
All TM marker genes tested were expressed by hTMCs and treatment with Dex led to a 35-fold increase in myocilin expression compared to vehicle control (p<0.05). Over the 96-hour period, subconfluent hTMCs contracted the collagen gels by 67.0% and contact-inhibited hTMCs by 62.7%, both comparable to HDFs at 71.3% contraction. HeLa contracted minimally (4.0%). Addition of Y-27632 significantly reduced contractability of all cell types except HeLa (p≤0.0001). These assays confirmed known characteristics of TMCs. The impact of UG treatment on contraction was not statistically significant at the concentrations tested. Of the UG receptors tested, LIMBR1L was detected in hTMCs, both by qPCR and IF.
Conclusions :
Under the conditions tested, no effect of UG on hTMC contractibility was found. Additional experiments, such as PLA2 activity and phagocytosis assays, are needed to understand the effect of UG on TM cell function. UG is thought to affect TM contraction via regulation of PLA2 activity. UG has also been shown to alter phagocytic activity of some cell types.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.