The vital role for nitric oxide in intraocular pressure homeostasis

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Abstract

Catalyzed by endothelial nitric oxide (NO) synthase (eNOS) activity, NO is a gaseous signaling molecule maintaining endothelial and cardiovascular homeostasis. Principally, NO regulates the contractility of vascular smooth muscle cells and permeability of endothelial cells in response to either biochemical or biomechanical cues. In the conventional outflow pathway of the eye, the smooth muscle-like trabecular meshwork (TM) cells and Schlemm's canal (SC) endothelium control aqueous humor outflow resistance, and therefore intraocular pressure (IOP). The mechanisms by which outflow resistance is regulated are complicated, but NO appears to be a key player as enhancement or inhibition of NO signaling dramatically affects outflow function; and polymorphisms in NOS3, the gene that encodes eNOS modifies the relation between various environmental exposures and glaucoma. Based upon a comprehensive review of past foundational studies, we present a model whereby NO controls a feedback signaling loop in the conventional outflow pathway that is sensitive to changes in IOP and its oscillations. Thus, upon IOP elevation, the outflow pathway tissues distend, and the SC lumen narrows resulting in increased SC endothelial shear stress and stretch. In response, SC cells upregulate the production of NO, relaxing neighboring TM cells and increasing permeability of SC's inner wall. These IOP-dependent changes in the outflow pathway tissues reduce the resistance to aqueous humor drainage and lower IOP, which, in turn, diminishes the biomechanical signaling on SC. Similar to cardiovascular pathogenesis, dysregulation of the eNOS/NO system leads to dysfunctional outflow regulation and ocular hypertension, eventually resulting in primary open-angle glaucoma.

Introduction

Despite the vast production of scientific work involving nitric oxide (NO) (more than 176,000 entries in PubMed to date), the discovery of NO as a molecule with physiological relevance is a relatively recent event (Moncada and Higgs, 2006; Murad, 2004). NO was previously known as a pollutant, but it was not until the late 1970's that the role of NO as a signaling molecule started to become clear. Furchgott and Zawadzki (1980) described the existence of a molecule that was produced by endothelial cells and relaxed smooth muscle. At that point, they called the "mediator" endothelium-derived relaxing factor (EDRF). Early work on EDRF revealed that acetylcholine, bradykinin and histamine all stimulated its production, and that EDRF acted via soluble guanylyl cyclase and was inhibited by hemoglobin and methylene blue (Furchgott et al., 1984; Ignarro et al., 1986). Some years earlier, Murad and colleagues were already working on the activity of guanylyl cyclase in response to NO (Arnold et al., 1977), independently of the EDRF studies.

NO meets all criteria for a prototypical gasotransmitter (Mustafa et al., 2009): it is light weight (MW = 30D); it is highly permeable with respect to lipid bilayers; it can be endogenously generated; it requires no exclusive cognate cell surface receptors (but it has several critical intracellular macromolecular targets); it has associated derivatives (e.g.: superoxide, nitrite, nitrate, nitrous oxide, peroxynitrite) that are critical to its function; and most importantly, it serves as a signaling molecule for a wide variety of essential physiologic functions including, as we will argue, intraocular pressure (IOP) regulation (Wang, 2018). There are other gasotransmitters, including carbon monoxide (Bucolo and Drago, 2011) and hydrogen sulfide (Han et al., 2019), but these will not be covered in this review because their role in ophthalmic physiology has not been studied as extensively as NO.

Leading to the identification of EDRF as NO, there were studies describing the short half-life of the molecule (Gryglewski et al., 1986) and the realization that EDRF was a free radical, due the observation that most of its inhibitors had redox properties that lead to the generation of superoxide (O2) (Moncada et al., 1986). It was in 1987 when Ignarro et al., 1987a, 1987b and Palmer et al. (1987) confirmed that EDRF was NO. Later, Palmer, Moncada and colleagues showed that L-arginine served as a substrate for NO production (Palmer et al., 1988), by NO synthase (NOS) (Moncada et al., 1989), which was isolated in 1990 (Bredt and Snyder, 1990). After 1990, the scientific production involving NO increased exponentially until it plateaued around the year 2000; since then, more than 7000 papers on NO are published every year. Due to the significance of these discoveries, NO was named “molecule of the year” in 1992 by Science (Culotta and Koshland, 1992) and Robert F. Furchgott, Louis J. Ignarro, and Ferid Murad won the Nobel Prize in physiology or medicine in 1998 ‘for their discoveries concerning NO as a signaling molecule in the cardiovascular system’ (Nicholls, 2019). Since then, NO has been shown to be a key mediator in most vascular beds in the body, including those in the eye.

In the first part of this review, we provide an overview of NO biology and its role in endothelial function and dysfunction. This will provide the necessary background to discuss the role of NO in aqueous humor dynamics, IOP regulation and pathology leading to primary open-angle glaucoma (POAG), which accounts for 75% of glaucoma globally and over 50% of glaucoma-related blindness (Quigley and Broman, 2006). Importantly, lowering IOP is the only efficacious means to slow POAG progression. Over the past decade, it has become clear that NO is a key regulator of IOP homeostasis within the conventional outflow pathway. In the second part of the review we provide a comprehensive examination of pioneering work in this realm, which together supports a novel mechanism by which NO modulates conventional outflow resistance to maintain IOP. Proper functioning of the conventional outflow pathway maintains IOP within a few mmHg throughout the lifetime of most people (Gabelt and Kaufman, 2005; Toris et al., 1999). However, its dysfunction is responsible for high IOP in POAG (Grant, 1951), which has motivated industry partners to develop technologies that target NO signaling to treat POAG.

Section snippets

Nitric oxide/nitric oxide synthase basics

NO is produced by NOS, which are a family of enzymes made of three isozymes transcribed from three different genes: neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3). nNOS and eNOS are constitutively expressed, while iNOS is “inducible”, being produced under pathological conditions as a mediator of the immune response. nNOS and eNOS are found in different cell types and tissues, but their names come from the cell types in which they were discovered and

Nitric oxide in the vasculature

NO is involved in many aspects of normal physiology depending on the resident cell/tissues. For example, NO participates in the regulation of vascular tone, platelet aggregation, inflammation, neurotransmission and immune response. Concerning IOP regulation, we will focus on the roles that NO plays on both vascular physiology and pathophysiology, especially on the effects of endothelial-derived NO in vascular tone and permeability.

Genetics

POAG is a common, complex disease with multiple target tissues including TM cells, SC endothelium, collector channels, ciliary body cells of several types, vascular endothelia that supply critical structures in the anterior and posterior segment, glial and other support cells and most importantly retinal ganglion cells (Fig. 4). Additionally, POAG is a highly heritable (Cuellar-Partida et al., 2016; Wang et al., 2017a) and polygenetic disease (Craig et al., 2020). Current genome-wide

Nitric oxide effects on IOP and outflow facility in different species

The importance of eNOS activity and NO bioavailability on IOP homeostasis and outflow facility has been studied by either inhibiting or stimulating eNOS function or NO supplementation. Shown in Table 1 is a compilation of studies that have assessed the effect of NO on IOP and/or outflow facility. The strategies used to study the effect of NO on outflow facility and IOP range from genetic modifications that result in overexpression or KO of eNOS to pharmacological treatments that inhibit eNOS or

Mechanisms of IOP mechanosensation and homeostasis in the conventional outflow pathway

Any model of IOP homeostasis requires a mechanism to sense and respond to changes in IOP. Mechanosensory mechanisms for IOP have been mainly attributed to IOP-induced stretch in the TM or shear stress due to circumferential aqueous humor flow in SC, although alternative models such as mechanosensitive nerve endings in the scleral spur (Tamm et al., 1994) have been proposed. As the bulk of outflow resistance generation lies within the outer TM, mechanisms for IOP mechanosensation within the TM

NO donating therapeutics

Given the hypothesized importance of NO in this IOP-sensitive feedback loop and that NO donors efficaciously lower IOP, targeting the NO pathway appears to be ideal for drug development to treat glaucoma. For example, in preclinical experiments topical or intravitreal administration of a number of different NO-donor molecules dramatically, but transiently decrease IOP in rabbits (Behar-Cohen et al., 1996; Carreiro et al., 2009; Kotikoski et al., 2002). Nipradilol, a beta-blocker with a nitroxy

Conclusions and future directions

In summary, NO plays a major role in regulation of IOP and outflow resistance, mediating physiological responses in the TM, SC, and distal vasculature. Shear stress is the main mechanical stimulus regulating NO production in the outflow pathway, and several factors influence how shear stress is detected or transduced such as TM stiffness, oscillations, and acute verses chronic shear stress. There is still a gap in knowledge as to how the SC cells sense and respond to mechanical stimuli such as

CRediT authorship contribution statement

Ester Reina-Torres: Investigation, Writing - original draft, Writing - review & editing. Michael L. De Ieso: Investigation, Writing - original draft, Writing - review & editing. Louis R. Pasquale: Investigation, Funding acquisition, Writing - original draft, Writing - review & editing. Darryl R. Overby: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. W. Daniel

Acknowledgements

The authors thank Nick Delamere for helpful discussions during the preparation of this review. Funding includes support from the National Eye Institute, EY022359 (WDS and DRO), EY028608 (WDS), EY005722 (WDS), EY015473 (LRP) and Research to Prevent Blindness Foundation (WDS).

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    1

    Authors contributed equally.

    2

    Percentage of work contributed by each author in the production of the manuscript is as follows: Ester Reina-Torres: 25%, Michael L. De Ieso: 25%, Louis R. Pasquale: 5%, Darryl R. Overby: 20%, W. Daniel Stamer: 25%.

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