Modulation of aqueous humor inflow and outflow in mammalian eyes

Abstract

Glaucoma is the leading cause of irreversible blindness worldwide. In principle, glaucoma could be treated by interrupting the pathogenesis of the disease or by neuroprotection of the retinal ganglion cells. However, currently, lowering intraocular pressure (IOP) remains the only effective intervention to slow the onset and progression of glaucomatous blindness. IOP can be lowered by reducing the rate of aqueous humor inflow, by reducing the flow resistance (increasing outflow facility) through the conventional (trabecular) outflow pathway, or by shunting outflow through the relatively pressure-insensitive uveoscleral outflow pathway. Diurnal variation of aqueous humor inflow has been well documented, with the rate falling by about a half in the early morning hours. In contrast, IOP does not display a proportional diurnal drop, which has been found to partly reflect a reduction in outflow facility. One major regulator of outflow facility is thought to be matrix metalloproteinase-9 and -2 (MMP-9 and MMP-2) secreted by the human trabecular meshwork (TM) cells. Since TM cells are subject to diurnal temperature oscillations arising from variations in core body temperature and eyelid closure during sleep, the effects of temperature oscillations on the activity of secreted MMP-9 and MMP-2 were investigated. The zymography results showed that the activity of MMP-9 and MMP-2 was increased with a temperature rise of 4°C. The changes were in the opposite direction when the temperature was lowered. Temperature cycling did not entrain clock gene expression, indicating that the MMP-release oscillations did not result from a circadian rhythm with a peripheral clock located in the TM cells. Heat shock transcription factor 1 (HSF1) inhibitor KNK437 reduced the MMP-9, but not MMP-2 temperature cycling-driven oscillations. In contrast, inhibition of TRPV1 or TRPM8 channels had no effects on temperature-driven MMP-9 and MMP-2 oscillations. Our results suggest that temperature cycling may alter MMP-9 and MMP-2 activity in the TM cells, potentially leading to extracellular matrix (ECM) remodeling and alteration of outflow resistance. Aqueous humor inflow is produced by the secretion of fluid and solutes across the bilayered ciliary epithelium. The solutes and fluid taken from the extracellular fluid by the pigmented ciliary epithelium (PE) is transferred to the non-pigmented ciliary epithelium (NPE). This fluid transfer is facilitated by intercellular gap junctions, where inflow can be inhibited by interrupting gap junctional communication. Various gap junction isoforms are expressed in the ciliary epithelium of different species, and their functional significance has not been completely elucidated. Gap junction connexins in the ciliary epithelium of pig, which is considered to be a good animal model for studying aqueous humor formation, have been characterized, using RT-PCR and Western blot (WB). Our results showed the presence of Cx43, Cx45, Cx47, Cx50 and Cx60 in porcine ciliary epithelium. Cx43 was found to be located primarily at the interface between PE and NPE cells by immunohistochemistry. The expression level of Cx43 was shown to be over 200-fold higher than that of the other connexins by qPCR. siRNA knockdown of Cx43 significantly reduced both mRNA and protein expression, as well as dye diffusion rate across isolated PE-NPE cell couplets. The results indicate that Cx43 provides a major conduit for fluid transfer between PE and NPE cells. Additionally, the effects of temperature and melatonin on the regulation of aqueous inflow were studied. Increasing the temperature by 4°C inhibited the short-circuit current (Isc) across porcine ciliary epithelium by half. The temperature-induced effect on Isc was reversible and repeatable. Increasing the incubation temperature by 4°C for 30 mins caused a reduction of Cx43 protein expression, while decreasing the temperature had an opposite effect in porcine ciliary epithelial cells. However, no changes were detected in the dye transfer rate across isolated PE-NPE cell couplets when the temperature was increased. The findings suggest that temperature alteration may have a direct effect on Cx43 expression without influencing gap junction permeability. In addition, melatonin was found to stimulate the transepithelial electrical measurements and facilitate the dye transfer across PE-NPE cell couplets. A pretreatment with MT1/MT2 antagonist luzindole did not inhibit melatonin-induced Isc stimulation, and MT3 antagonist prazosin blocked its stimulation completely. The findings suggest that melatonin may facilitate the solute and fluid movement from PE to NPE cells, leading to an increase in transepithelial secretion, potentially via a putative MT3 receptor. Whether or not it contributes to the observed diurnal changes in aqueous humor flow and IOP awaits further investigation.