Role of aquaporin-1 in trabecular meshwork cell homeostasis during mechanical strain

Abstract

Aquaporin-1 (AQP1) channels are expressed by trabecular meshwork (TM) and Schlemm's canal cells of the conventional outflow pathway where fluid movement is predominantly paracellular, suggesting a non-canonical role for AQP1. We hypothesized that AQP1 functions to protect TM cells during periods of mechanical strain. To test this idea, primary cultures of confluent human TM cells on Bioflex membranes were exposed to static and cyclic stretch for 8 and 24 h using the Flexcell system. AQP1 expression in TM cells was assessed by SDS-PAGE and Western blot using anti-AQP1 IgGs. AQP1 protein bands were analyzed using densitometry and normalized to β-actin expression. Cell damage was monitored by measuring lactate dehydrogenase (LDH) and histone deacetylase appearance in conditioned media. Recombinant expression of AQP1 in TM cell cultures was facilitated by transduction with adenovirus. Results show that AQP1 expression significantly increased 2-fold with 10% static stretch and 3.5-fold with 20% static stretch at 8 h (n = 4, p < 0.05) and 24 h (n = 6, p < 0.05). While histone deacetylase levels were unaffected by treatments, release of LDH from TM cells was the most profound at the 20% static stretch level (n = 4, p < 0.05). Significantly, cells were refractory to the 20% static stretch level when AQP1 expression was increased to near tissue levels. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r² = 0.7780). Taken together, AQP1 in human TM appears to serve a protective role by facilitating improved cell viability during conditions of mechanical strain.

Introduction

Glaucoma is the second leading cause of blindness and affects approximately 3 million people in the United States alone (Quigley, 1996). Primary open angle glaucoma is the most common form and is often characterized by an increase in intraocular pressure (IOP). Despite visibly unobstructed conventional outflow tissues, an increase in resistance through the conventional outflow pathway is likely responsible for IOP elevation (Grant, 1963).

The conventional outflow pathway consists of the trabecular meshwork (TM) and Schlemm's canal (SC), and is part of a dynamic environment subject to multiple forms of stress including mechanical strain. Sources of daily mechanical strain in the conventional outflow pathway include intraocular pressure, fluid flow, and contractile activity of surrounding tissues, such as the ciliary muscle. Intraocular pressure and fluid flow are influenced by intraocular processes such as aqueous humor production and drainage (Kaufman, 1984), as well as extraocular processes such as heart beat, eye movement, and blinking (Coleman and Trokel, 1969). The TM is subject to added sources of strain as mechanical forces stretch it from Schwalbe's line to the Scleral spur, and inward towards the SC lumen (Johnstone and Grant, 1973). As a result, the TM stretches not only in accordance with changing pressure gradients and fluid movement, but also in conjunction with ciliary muscle contraction (Wiederholt et al., 2000).

Studies using whole eyes, anterior segments, and isolated TM cultures have demonstrated a range of response mechanisms to mechanical strain. Experiments in eyes of rhesus monkeys and humans revealed reversible structural changes in TM tissues following increases in pressure (Johnstone and Grant, 1973). Perfusion studies using anterior segments of human and porcine eyes reported an increase in outflow resistance (Brubaker, 1975) and a decrease in outflow facility following applied cyclic pressure, suggesting pressure oscillations can induce responses in tissue (Ramos and Stamer, 2008). Investigators have also looked at alterations in cell morphology and actin reorganization following applied mechanical strain to cultured human TM cells (Brubaker, 1975, Epstein and Rohen, 1991, Mitton et al., 1997, Tumminia et al., 1998). Moreover, changes in gene expression have been observed for multiple proteins including myocilin, interleukin factor-6, and matrix metalloproteinases, following applied mechanical strain in TM cultures (Borras et al., 2002, Bradley et al., 2001, Bradley et al., 2003, Liton et al., 2005, Tamm et al., 1999, Vittal et al., 2005). Regulators of transport mechanisms, commonly associated with cell homeostasis are also influenced by mechanical strain in the TM and other tissues (Gasull et al., 2003, Yuan et al., 2007). Interestingly, AQP4 has been shown to facilitate increased water flux between muscle tissue and the blood during increased physical activity, demonstrating a novel role for aquaporin channels during times of mechanical strain (Frigeri et al., 2001, Frigeri et al., 2004).

Aquaporin water channels serve to increase water permeability of cells and facilitate transcellular water movement in the kidney, lung, and the eye (amongst others); tissues whose proper functioning depends upon efficient water movement. In the conventional outflow pathway, AQP1 has been shown to be expressed in both TM tissue and isolated cultures, though the role of AQP1 in the TM is currently unknown (Hamann et al., 1998, Stamer et al., 1994, Stamer et al., 1995, Stamer et al., 2008). Since fluid movement through the TM is primarily paracellular, the presence of AQP1 may fulfill a need other than transcellular water movement, particularly in the uveal meshwork where intertrabecular spaces are large. In fact, recent studies have demonstrated that AQP1 does not appear important for bulk fluid movement through the conventional outflow pathway (Stamer et al., 2008). It is currently unclear why robust expression of a water channel protein in the TM would be necessary if fluid movement does not require AQP1 expression. Based upon the unique biomechanical environment of the conventional pathway and recent reports of the role of aquaporins during periods of mechanical strain, we hypothesize that AQP1 functions to support TM cell homeostasis during times of mechanical strain.

To test our hypothesis we administered a static and cyclic mechanical stretch to cultured human TM cells and evaluated changes in AQP1 expression and TM cell viability. Our results demonstrate that mechanical strain results in a significant increase in AQP1 expression. To further evaluate the role of AQP1 during mechanical strain we used adenovirus encoding AQP1 to restore AQP1 expression in TM cell cultures to levels closer to those observed in TM tissue. We found that increased expression of AQP1 during static mechanical strain reduced measures of cell damage. Based on the current study, our data suggest that AQP1 in human trabecular meshwork serves a protective role by facilitating improved cell viability during conditions of mechanical strain.