TRPV4 regulates intraocular pressure through trabecular meshwork contractility and fibrosis

ABSTRACT

Intraocular pressure (IOP) is dynamically regulated by the contractility and viscoelasticity of the trabecular meshwork (TM). Two recent studies identified the polymodal cation channel TRPV4 as a central mechanosensor that integrates mechanical, biochemical, and circadian signals to set the IOP levels. Pharmacological TRPV4 inhibition, global Trpv4 knockout, and conditional deletion of Trpv4 attenuated pathological ocular hypertension induced by corticosteroids, TGFβ2, or angle occlusion, as well as physiological nocturnal IOP elevation. Conversely, the selective TRPV4 agonist GSK1016790A raised IOP when injected intracamerally but lowered it when applied topically, indicating compartment-specific action. TRPV4 activation induced actomyosin contractility and ECM deposition in cultured TM cells and increased outflow resistance in biomimetic 3D scaffolds and hydrogels, with the impact reversed by TRPV4 inhibition and gene deletion. TGFβ2 strongly upregulated transcription and functional expression of TRPV4, revealing a feed-forward fibrotic loop that may contribute to myofibroblast transdifferentiation of the stressed TM. Collectively, these findings established TRPV4 as an essential mediator of TM contractility, stiffness, and IOP homeostasis. Its expression in key pressure-regulating tissues (TM, Schlemm’s canal, ciliary body, and ciliary muscle) positions the channel as a convergence point for diverse glaucoma risk factors that regulate aqueous fluid production and drainage, and thus as a promising therapeutic target to lower IOP without global disruption of actin polymerization.

Introduction

From the mechanobiologist’s perspective, the rich tapestry of effects exerted by intraocular pressure (IOP) on ontogenetically, structurally, and functionally diverse tissues of the vertebrate eye represents a continual source of fascination. Required to maintain the shape of the eye globe, IOP is tightly controlled over the lifetime of (mouse or human) individuals [1], with chronic decreases and increases associated with vision loss due to optic nerve damage (glaucoma), neuroinflammation, choroidal detachment, and macular edema. Because intraocular pressure (IOP) is the only modifiable risk factor for glaucoma, effective therapy necessitates an in-depth understanding of molecular mechanisms that subserve the induction and maintenance of ocular hypertension.

Chronic IOP elevations arise predominantly from increased resistance within the conventional outflow pathway, governed by the trabecular meshwork (TM), to the outflow of aqueous humor into lymphatic-vascular circulation [2]. Thishighly mechanosensitive, contractile, and phagocytic tissue is located at the iridocorneal angle [2–4] is and populated by cells that respond to mechanical stretch, corticosteroids, and TGFβ2 with enhanced contractility and deposition of extracellular matrix (ECM), which in turn stiffens the outflow pathway and increases its resistance to paracellular fluid flow [2–4]. Circadian factors, presumably under the control of the suprachiasmatic nucleus and the superior cervical ganglion [5], reversibly elevate IOP at night and lower it during the day. IOP elevations increase the trabecular outflow resistance [6] by stretching ECM beams that traverse the TM and thereby inducing contraction of resident cells. This mechanism has been explored in ex vivo experiments of stretch-induced contractility of intact TM tissue [7], Rho signaling and extracellular matrix (ECM) secretion and degradation [8]. Actin depolymerizing drugs (e.g., latrunculins and cytochalasin D), Rho kinase inhibitors (e.g., netarsudil, Y-2723) and ECM-degrading enzymes [5,9–11] lower TM resistance to fluid flow in vitro and in vivo, pointing at actomyosin contractility and ECM fibrosis as final common mechanisms of ocular hypertension (OHT). A major remaining challenge has been to delineate the molecular mechanisms that mediate the coupling between mechanical (pressure, stretch, shear) forces, chemical (TGFβ2, steroids), and circadian factors, cytoskeletal dynamics, and fibrotic remodeling. The recent reports by Rudzitis et al. [12] and Redmon et al. [13] combined pharmacological strategies with global and conditional knockout mouse models and contractility studies in cultured TM cells to converge on the same conclusion: TRPV4 activity within the anterior eye is dispensable for baseline IOP but obligatory for physiological (circadian) and pathological ocular hypertension.

TRPV4 structure, mechanosensitivity, and trabecular functions

TRPV4, a polymodal, tetrameric nonselective cation channel with P_Ca/P_Na ~10, is composed of six transmembrane domains with the pore between TM5 and TM6 [14]. Its N-terminus includes a proline-rich domain associated with volume sensitivity [15], an intrinsically disordered region (IDR) that may bind phosphatidylinositol-4,5-biphosphate (PIP2), and six AR (ankyrin repeat) domains implicated in temperature sensing and oligomerization; the GTPase RhoA, recently shown to bind AR2-AR5, dissociates from the channel upon activation [16,17]. The C-terminus includes an amphipathic TRP domain implicated in pore gating and a CaM binding site and may bind inositol triphosphate calcium release channels in subsurface ER cisternae, and a PDZ-binding-like motif that regulates interaction with YAP/TAZ proteins [16]. The overall structure and homology to cognate vanilloid isoforms predict a propensity for protein–protein interactions, posttranslational modifications and modulation by Ca²⁺, highlighting the cell type- and context-dependence of TRPV4 activation and function [18,19]. Originally described as an osmosensor, TRPV4 may also be activated by matrix stiffness, cyclic strain, compression, shear stress, moderate heat, UV light, and numerous lipid mediators [16,20–22]. Its ocular expression includes endothelia (vascular, lymphatic, corneal), epithelia (corneal, ciliary body, retinal pigment, scleral, and lens), retinal ganglion cells, and glia [15,19,23,24]. TRPV4-deficient mice show normal IOP and normal responsiveness to light without overt structural phenotypes [13,25]; however, TRPV4 channelopathies have been linked to sensorimotor neuropathies with phenotypes ranging from mild to neonatal lethality [26], TRPV4-related SNPs were reported in patients with normal tension glaucoma [27], and loss-of-function mutations were associated with retinal degeneration [28].

Mouse and human TM cells utilize TRPV4 to sense and transduce membrane and ECM stretch, shear, temperature, and swelling [29–34]. Its slow activation kinetics (~sec; [31]) presumably reflects intermediation by the canonical phospholipase A2 cascade that culminates in production of epoxyeicosatrienoic acids as the final activators of the pore [23,29,30] that is additionally modulated by cholesterol and lipid rafts [35]. TRPV4 mechanosensitivity predicts functions in pressure-dependent modulation of conventional outflow resistance [6] yet studies conducted over the past 15 years disagree about whether TRPV4-mediated Ca²⁺ influx facilitates or impedes fluid drainage and what its role in IOP homeostasis might be. TRPV4 activation within the TM has been suggested to lower IOP via phosphoinositide signaling [36], release of polyunsaturated fatty acids [32] and eNOS activation [33,36], and, conversely, to elevate IOP through cytoskeletal upregulation and fibrosis [8,29]. Investigations of the TRPV4-dependence of IOP homeostasis in global Trpv4 knockout mice similarly yielded conflicting results [29,32,33,36], with the added lack of clarity from pharmacological studies, which suggested that IOP can be lowered by systemic and topical administration of either TRPV4 agonists or antagonists [29,33,36]. It has been unclear whether the disparate outcomes reflect activation of TRPV4 pools in different cell types, species specificity, differences in OHT induction and/or involvement of non-trabecular mechanisms. The reports addressed by the Commentary [12,37] tested these possibilities across multiple models of (physiological and pathological) ocular hypertension, by studying outflow pathway-specific and global Trpv4 knockout mice, by taking advantage of multiple agonist/antagonist application approaches, and by evaluating the TRPV4-dependence of TM contractility in vitro using complementary biomimetic assays of trabecular outflow.

TRPV4 is required for physiological and pathological ocular hypertension

The two studies converged on the same conclusion: TRPV4 activity is dispensable for baseline IOP but obligatory for physiological (circadian) and pathological ocular hypertension. Selective TRPV4 antagonists (HC067047 and GSK2193874) did not affect normotension but lowered IOP in mouse models of TGFβ–2, angle occlusion- and steroid-induced OHT as well as during the nocturnal phase of the circadian cycle (Figure 1(A)) The attenuation of OHT in global and conditional Trpv4 knockout mice (Figure 1(B)) confirmed that TRPV4 channels are necessary for OHT maintenance while additionally indicating functional contributions from trabecular and nontrabecular TRPV4 pools. Intracameral injection of the agonist GSK1016790A lowered IOP while topical corneal administration increased it – likely via TRPV4- and Pnx1-dependent release of purines from corneal epithelial and endothelial cells [19]. Unexpectedly, TRPV4 inhibitors also lowered IOP during the nocturnal hypertensive IOP phase [37]; the mechanisms that reversibly elevate IOP at night remain unknown but could involve modulation of glucocorticoid production, melatonin, norepinephrine signaling [5] and/or changes in TRPV4 expression [38].

Figure 1.

(A) Time course of IOP lowering following intracameral injection of the antagonist HC067047 in 3 mouse models of ocular hypertension (DEX (N = 7), TGFβ2 (N = 5), angle occlusion (N = 9)). (B) Time course of OHT in wild type (N = 8), conditional KO (Mgp^CreTrpv4^fl/fl) (N = 9) and global Trpv4^-/- (gKO) (N = 6) animals. See methods in [12,13] for experimental details.

It is important to note that TM cells utilize Piezo1 [31], TREK-1 [39] and TRPM4 [40] channels in addition to TRPV4 to respond to mechanical stressors. Intracameral inhibition of Piezo1 suppressed the outflow facility [31] while TREK-1 activation increased it [41] to lower IOP in hypertensive eyes [13,29,42]. Taken together, these findings suggest that mechanosensitivity of the conventional pathway operates around a dynamic setpoint set by opposing fluxes of Na⁺, K⁺ and Ca²⁺ ions [2,43]; the osmoregulatory setpoint that controls the TM cell volume additionally involves Cl⁻ flux through VRAC and ANO6 channels [34].

TRPV4 controls TM contractility and trabecular outflow resistance

To establish the role of TRPV4 in the regulation of trabecular outflow resistance apart from the involvement of inflow, secondary outflow (ciliary muscle) and Schlemm’s canal components, the studies took advantage of complementary 3D biomimetic systems (porous silicone nanoscaffolds and collagen I hydrogels). Consistent with in vivo observations, TRPV4 activation increased and inhibition decreased, the resistance of bioengineered nanoscaffolds to pressurized fluid flow [13,29]. Furthermore, the agonist GSK1016790A induced TM contraction while the antagonist blocked TGFβ2 -evoked contractility in hydrogels that simulated the in situ 3D biomechanical environment [12,44]. TGFβ2 upregulated the transcription and functional expression of TRPV4 (and PIEZO1) [12] to presumably amplify mechanosensitivity of s the positive feedback loop that links IOP to trabecular stretch, contractility, stiffness, and flow resistance.

Proposed mechanism for TRPV4 signaling within the TM

These findings place TRPV4 mechanotransduction upstream from RhoA/ROCK signaling, Ca²⁺-induced cytoskeletal reinforcement and ECM fibrosis that underpin paratensile TM force generation and myogenic-like tone that controls aqueous humor outflow and thereby IOP. Mechanical stretch, steroids and TGFβ2 converge on TRPV4 to drive short-term contractility and long-term myofibroblast transdifferentiation by shifting the IOP setpoint toward a hypertensive contractile state (Figure 2). At the molecular level, TRPV4-mediated Ca²⁺ influx activated by tensile and compressive forces [45] facilitates Ca²⁺-Induced Ca²⁺ release [29], formation of stress fibers [8,29], reinforcement of integrin-based cell-ECM contacts, tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin and vinculin, and translocation of activated zyxin into stress fibers [8] through coordinated activity of the RhoA GTPase, a quasi-subunit of TRPV4 [17] that is induced within 15’ of mechanical stretch [8]. TRPV4 activity reduces the cellular pool of G-actin to augment force production through increased actin polymerization and actomyosin contractility, and indirectly by promoting expression of profibrotic genes that underpin the epithelial-mesenchymal transition (e.g. SNAIL1 and ACTA2). TGFβ hijacks these pathways by inducing TRPV4 overexpression [12] and driving SMAD- and RhoA-dependent hypercontractility, matrix stiffness and fibrotic remodeling.

Figure 2.

Schematic representation of TRPV4 signaling as a final common pathway in OHT. TGFβ2 and glucocorticosteroids stimulate fibrosis and contractility through canonical (SMAD) and noncanonical (RhoA) mechanisms and modulation of gene expression. Activated TRPV4 elevates IOP by promoting ECM deposition and actomyosin contractility, presumably in collaboration with integrin signaling.

TRPV4 signaling within the anterior eye

The two studies [12,13] suggest the overall effect of topically, intracamerally and systemically administered TRPV4 inhibitors is to lower IOP in hypertensive eyes, with the TM as a principal effector of TRPV4-dependent IOP regulation. It is important to note that TRPV4 could play additional homeostatic and pathological functions by regulating fluid secretion from nonpigmented epithelial cells of the ciliary body [24], contractility of the ciliary muscle [19], and permeability of endothelial cells that line the canal of Schlemm . The role of TRPV4 activity in Schlemm’s canal pressure sensing remains unclear, as the TRPV4 agonist GSK1016790A facilitates formation of paracellular “pores” within Schlemm’s canal cells while also increasing their contractility and stiffness [46]. Our finding that endothelial cells respond to TRPV4 activation with dissolution of adherens junctions and decreased barrier flow resistance [47] suggests trabecular vs. endothelial TRPV4 channels culd have opposing functions on flow regulation. TRPV4 channels thus exemplify the complexity of mechano-biochemical interactions that subserve IOP homeostasis iunder physiological and pathological conditions.

Therapeutic and conceptual implications

1. TRPV4 inhibition lowers IOP in all studied models of ocular hypertension

2. Topical TRPV4 agonists lower IOP by targeting corneal rather than TM channels, intracameral agonists elevate IOP by augmenting the myogenic-like tone of TM and ciliary muscle cells.

3. Lack of additivity in TGFβ2 and nocturnal IOP [12] indicates TRPV4 represents the final common mechanism of physiological and pathological ocular hypertension .

4. TRPV4 sets the IOP setpoint together with PIEZO1 and TREK-1 channels [31,37,48].

5. Pathology influences ocular mechanochannel expression and function [12,41], which should be taken into account by therapeutic interventions.

6. TRPV4 inhibition may offer superior safety compared to broad actin-depolymerizing or Rho kinase-inhibiting strategies, as it preserves basal cytoskeletal integrity and obviates hyperemia.

Funding Statement

This work was supported by the National Institutes of Health [T32EY024234, R01EY022076, R01EY034086, R01EY1031817, P30EY014800], Crandall Glaucoma Initiative, Stauss-Rankin Foundation and Unrestricted Grants from Research to Prevent Blindness to the Department of Ophthalmology at the University of Utah.

Disclosure statement

D.K. is the CFO for TMClear.

Data availability statement

The m/s does not include primary data.