Abstract
Purpose :
The presence of uveolymphatic pathways were recently discovered and may contribute to drainage of aqueous humor and regulation of intraocular pressure (IOP). Bimatoprost is a prostaglandin-analog commonly used to reduce IOP. Prior work with topical bimatoprost in monkey demonstrated partially endothelial lined passages within the ciliary body stroma. We hypothesized that these passages are lymphatic in nature and human ciliary body smooth muscle (CBSM) cells are the source of the lymphangiogenic proteins.
Methods :
CBSM cells were dissected and cultured from the anterior segment of four human donors ages 60, 62, 73, and 76. CBSM cells were treated with vehicle control (0.001% ethanol), 3.3, 10, 25.7, or 257 nM of bimatoprost or bimatoprost free acid. Immunoblot analysis was performed on cell lysates to determine the effect of drug treatment on protein expression for the lymphatic markers: LYVE-1, PROX-1, TIE-1, ANG-1, ANG-2, and NRP-2. Fold-change as compared to vehicle control was quantified and two-tailed paired t-tests were used for statistical analysis.
Results :
Bimatoprost treated CBSM cells demonstrated a 33 ± 7% decrease in NRP-2 expression (n=3; p=0.048) at 10 nM, while no dose-dependent trend was observed for LYVE-1 (n=2), TIE-1 (n=3), or PROX-1 (n=3). ANG-1 (n=2) and ANG-2 (n=1) exhibited a dose-dependent although non-statistically significant increase in protein expression with a maximal increase of 56% and 64% for ANG-1 and ANG-2, respectively. Bimatoprost free acid treated CBSM cells (n=1 for each target) exhibited a dose-dependent decrease in NRP-2, no dose-dependent trend in LYVE-1, PROX-1, or ANG-2, and a dose-dependent increasing trend in TIE-1 and ANG-1.
Conclusions :
Our study confirmed the expression of lymphatic markers by human CBSM cells, and that bimatoprost and bimatoprost free acid had differential effects on lymphatic gene expression in CBSM. The significant decrease in NRP-2 at 10 nM of bimatoprost may be due to changes in receptor trafficking since NRP-2 is a co-receptor for VEGFR-3. Increases in TIE-1, ANG-1 and ANG-2 may indicate development of lymphatics in response to drug treatment. Experiments are ongoing to better characterize this trend.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.