Lipids and Lipid Regulators in Intraocular Pressure Homeostasis

Abstract

Increased intraocular pressure is the strongest correlated modifiable risk factor for developing primary open angle glaucoma (POAG). Lipids have long been known to be a major constituent of aqueous humor. Lipid mediators, prostaglandins for example, are the first-line treatment for glaucoma. Innovative technologies have made the investigation of lipids in small quantities possible, and interest in identifying lipids as new pharmacological targets has grown in ophthalmology. There is expanding evidence to suggest that lipids and their active metabolites play a role in POAG pathophysiology, as differences between control and diseased eyes have now been demonstrated. The role of these differences is yet to be determined and is the subject of this review.

Introduction

Lipids are an essential macromolecule to biology, conferring structural, communicational, and energetic functions to a cell. [1] Lipids are the focus of this review primarily for their relevance to intracellular signaling and the maintenance of cellular structure in the trabecular meshwork (TM) outflow pathway. The TM outflow pathway regulates aqueous humor (AH) drainage.[2] Dysregulation of AH drainage due to TM dysfunction plays a central part in elevating intraocular pressure (IOP). Thus, elevation in IOP is the largest risk factor for developing primary open-angle glaucoma (POAG).[3] Additionally, we can speculate that AH lipids can alter the extracellular signals to the TM and therefore modify AH drainage leading to IOP dysregulation.

Brief Review of Glaucoma and Elevated Intraocular Pressure

Glaucoma is the most common cause of irreversible vision loss worldwide, and second only to age-related macular degeneration (AMD) in the United States.[4] Progressive visual field loss is the hallmark of glaucoma, reflected physiologically by the loss of the inner-most cell layer of the retina, namely retinal ganglion cells (RGCs) and their axons. Elevated IOP is the most highly correlated and modifiable risk factor.

It has been previously shown that increasing the rigidity of the TM will decrease the outflow of AH, resulting in elevated IOP.[5, 6] In glaucomatous eyes, the TM has greater stiffness, and the interface of the TM and Schlemm’s Canal (SC) has decreased elasticity.[7] POAG patients have interspersed areas of low TM outflow with higher stiffness and rigidity compared with high-flow areas.[8–10] Apart from elevated IOP, additional risk factors most especially include age, but also genetics. Additional research suggests that long ocular exposures to oxidative damage may also play a role.[11] Hyperlipidemia, elevated blood pressure, increased body mass index, metabolic syndrome, and other insulin-resistance-related features also appear to be positively correlated with elevated IOP.[12, 13]

Lipids in the Aqueous Humor and Signaling in TM

Since the aqueous chamber is largely avascular with AH providing nutrition to these structures, reviewing the lipid profiles of the AH and their effects is critical. The range of lipid species found in the aqueous chamber (AC) is vast, including species of fatty acyls (FA), glycerolipids (GL), glycerophospholipids (GPL), sphingolipids (SP), sterol lipids (ST), and saccharolipids (SL) (Figure 1). [14, 15] Other studies also concluded that GPL species were much more common than SP lipids, together constituting the most common lipids in the aqueous chamber.[16] Not only is there a difference in the levels of common lipids found in normal and POAG AH, but there are also unique species of lipids found in each state, as well as when comparing AH of controls with other controls, as well as POAG AH with other types of glaucoma AH samples, suggesting intergroup variance.[17] This change in the lipidome of the AH also occurs with other pathologies.[14]

Figure 1:

A Venn Diagram visualizing the general overlaps of the 8 LIPID MAPS categories. Not to size or scale, but to help the reader understand how some species of lipids can be considered in many categories. Sphingolipids (SPL) are present twice due to its relationships with various other lipid categories. Fatty Acyls (FA), Glycerolipids (GL), Glycerophospholipids (GPL), Prenol Lipids (PL), Saccharolipids (SL), Sphingolipids (SPL), Sterol Lipids (STL), Polyketides (PK). Created using BioRender.com.

Fatty acyls (FA) have been long documented to be present in the aqueous chamber [18] and the various species documented are broad.[14, 19] Some of the relevant FAs found in the aqueous chamber include various polyunsaturated fatty acids (PUFAs). PUFAs of note include arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA)[19]; DHA and EPA are omega-3 fatty acids while AA is an omega-6 fatty acid.

Omega fatty acids are a prominent class of fatty acyls due to their relevance in ocular biology.[15] One specific example is AA which is cleaved from the membrane by phospholipase 2 and metabolized by COX1 and COX2 creating prostacyclin, prostaglandins, and thromboxanes. [Figure 2] [20] [1] Leukotrienes are cousins to these lipid mediators and are also derived from arachidonic acid (AA), though they are synthesized through the lipoxygenase pathway. Together, these metabolites, including many other derivatives of arachidonic acid, are known as eicosanoids. In general, prostacyclin and prostaglandin molecules generate inflammation and induce pain signaling.

Figure 2:

Flow chart representing the various pathways that polyunsaturated fats (PUFAs) can be used to create various active lipid metabolites. Arachidonic Acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA). Created using BioRender.com.

Although AA is a crucial substrate for producing various bioactive signaling molecules in the body, it has not been identified as having a strong signaling role by itself. Despite this being the case, AA is elevated in the AH of glaucoma patients, along with its common synthesis byproduct, lyso-platelet activating factor (PAF), a precursor to PAF. The increase in lyso-PAF appears to be a negative change, as PAF is known to be a crucial part of ocular inflammation and glaucoma, as well as promoting ocular hypertension. This is potentially contributing to RGC damage via increased glutamate release and subsequent excitotoxicity.[18, 21, 22] Counterintuitively, levels of prostaglandins and leukotrienes, two of the major classes of AA derivatives, were not found to increase in the AH of POAG patients despite the increase in AA, suggesting a hitherto unelucidated reason for this change.[19] Additional work has also found that there is a decreased concentration of cyclooxygenase −1 and −2 (COX1 and COX2) in the non-pigmented ciliary epithelial cells in glaucomatous eyes which are the essential enzymes in prostaglandin synthesis.[23]

Beyond the domain of lipids lies the array of carrier proteins that expand lipid mobility. Apolipoproteins are a class of carrier proteins, which aid the transport of hydrophobic lipids through the vasculature in high- and low-density lipoprotein clusters (HDL, LDL). The proteins of HDL are Apolipoprotein A-I (Apo A), Apolipoprotein E (ApoE), and Apolipoprotein J (ApoJ), also known as clusterin.[24] This complex (HDL) is generally considered protective against cardiovascular disease.[24] Interestingly, clusterin also acts as a secretory chaperone protein. Of particular note in the TM is ApoJ, which has anti-inflammatory properties and has been noted to decrease in macular degeneration [24] but changes in its level are not fully elucidated in glaucoma pathophysiology. Correspondingly, there is evidence for its benefits in reducing IOP by an unknown mechanism, which is actively being investigated by the lab.[25]

Lipoprotein particles and their associated apolipoproteins (Apo) have been implicated in glaucoma, with increased risk being associated with higher levels of Apo B, increased Apo B/Apo A1 ratio, and increased LDL-cholesterol.[12] HDL-cholesterol appears to be somewhat occluded in its specific role, with studies disagreeing on its impact on IOP.[12] Decades of studies have suggested that race, specifically those of African and Latino/Hispanic heritage, may play a role in increased glaucoma risk and poorer visual outcomes.[26, 27] These differences are likely due to a combination of genetic, environmental, and socioeconomic factors, the latter which are found at higher rates in the aforementioned communities.[28]

Currently, the prostaglandin (PG) analogs of PGF2α and PGE2 are the first-line treatment for POAG.[29] Prostaglandins generally decrease IOP by mainly increasing the outflow of AH through the uveoscleral pathway.[30–35] Because PGF2α and PGE2 are so well studied and already in use as IOP-lowering drops with minimal negative side effects, research into alternative prostaglandins has not been pursued to a large degree.[35] Importantly, combinatorial drugs including prostaglandins have been tested for many years now. Examples include Rho kinase inhibitors and nitric oxide donating prostaglandins.[36–38]

DHA is an important building block of cell membranes. Because of the polyunsaturated nature of DHA, the integration of these lipids in cell membranes shifts the phase behavior toward a liquid state.[39] It has been previously shown that increasing the rigidity of the TM will decrease the outflow of AH, resulting in elevated IOP [5, 6] suggesting the importance of maintaining the correct fluidity of cell membranes in the AH outflow system.[40] In glaucomatous eyes, the TM has greater stiffness, and the interface of the TM and SC has decreased elasticity.[7, 41–43] Supplementation with omega-3 fatty acids decreases IOP, potentially highlighting this effect and the importance of these lipids within the AH.[44, 45] However, although the presence of omega fatty acids has been confirmed in the AH, any difference between the concentrations of control and glaucomatous eyes has not been established.[19] Another study showing the presence of omega-3 fatty acids in AH samples also found that their derivatives, deemed resolvins and protectins, play anti-inflammatory and even neuroprotective roles.[45]

Glycerophospholipids (GPL) are the main component of cell membranes. It has been shown previously that phosphocholines influence the outflow of the TM via the membrane dynamics of TM cells.[46–48] GPLs have been found to decrease the AH of glaucomatous human eyes.[49] Certain species of GPLs have been found to have beneficial effects on IOP regulation in mouse models.[16]

In DBA/2J mice, sphingolipids were found to be decreased in ocular hypertension when compared to control eyes.[48] However, other studies found the opposite in the same species of mice.[16] This same conundrum also exists within studies measuring sphingolipids in human AH.[16, 47, 50] Sphingolipids are involved with volume-sensitive chloride currents in myocytes[51] and similar mechanisms involving these chloride channels have been implicated in TM cell control.[52, 53] A specific metabolite of sphingolipids, named sphingosine-1-phosphate (SP1), has been shown to decrease AH outflow in human, porcine, and mouse eyes.[54–56]

One significant change found in POAG AH when compared to control AH is the concentration of cholesteryl esters, which was found to increase substantially.[50] Concerning cholesterol, statin use has been found to decrease the risk for POAG.[57]

A rising area of investigation is that of exosomes and their role in eye health.[58–61] Exosomes are extremely small, bi-layer membrane-bound micro-vesicles ranging in size between 50 – 160 nm and are ubiquitously produced by all living cells.[62] Exosomes are known to play powerful paracrine and autocrine signaling roles in the body. Differing from other lipid signals, exosomes contain several signal molecules including specialized lipid mediators, transcription factors, micro-RNA (miRNA), messenger RNA (mRNA), small interfering RNA (siRNA), DNA, and proteins.[63, 64] Exosomes are active in IOP regulation and the retina, RPE, and other ocular tissues.[58–61, 65] Exosomes appear to be especially active in cellular stress, potentially acting in a protective and restorative way to help re-regulate the IOP system. Interestingly, these restorative effects of exosomes produced by nonpigmented ciliary epithelium (NPCE) do not exist when in a non-stressed state but regain this function when exposed to a stressed state.[58, 60, 64] Exosomes produced by NPCE in POAG patients are not only smaller,[66] they are more numerous,[67, 68] have different lipid profiles, decreased ability to load extracellular matrix recycling proteins, and have decreased ability to bind to fibronectin. This fibronectin-binding ability also appears to increase in exosomes derived from healthy NPCE which undergo stress. Increased exosome density in the context of POAG was correlated with more severe visual field loss.[67]

The Trabecular Meshwork Lipids

Though the AH and TM have a similar amount of phospholipid species, only a dozen or so are common between them in healthy controls.[16] Sphingolipids are also present in TM cells, but are less common in comparison, while common species are also much fewer in number. The trend of fewer phospholipids and sphingolipids in the AH of POAG patients has also been proposed in TM cells.

However, recent research has also demonstrated the inverse, finding a general increase in lipid species of in-vitro human TM cells in response to mechanical stress, subsequently increasing the stiffness of TM.[69] One of many identified pathways proposes that cellular change occurs following activation of the sterol regulatory element binding proteins (SREBPs).[70] These proteins are transcription factors that bind to the sterol response element and upregulate lipogenic pathways in the cell.

Lysophospholipids have also been described in the TM and AH of both control and POAG patients, decreasing in diseased eyes.[71] Interestingly, higher concentrations of lysophospholipids are related to decreased AH outflow with elevated levels in POAG patients.[56, 72, 73] Their effects on TM cells include Rho GTPase activation, inducing acto-myosin-based cellular contraction, and increasing ECM protein expression.

Endocannabinoids are a species of lipid mediators derived from AA. Their IOP-lowering nature was first described in 1971.[74] After this initial discovery, many subsequent investigations into other natural or synthetic cannabinoids have corroborated this effect in humans. This was confirmed by inhibiting the IOP-lowering effects of endocannabinoids using a CB1 antagonist.[75]

The most common endocannabinoids are arachidonylethanolamide or anandamide (AEA) and 2-arachidonoylglycerol (2-AG). Interestingly, AEA can be modified by COX-2 to synthesize prostaglandin-ethanolamides (prostamides) and prostaglandin glyceryl esters, which are analogous to PGE2 and PGF2a.[76] Investigations into the changes of endocannabinoids in glaucoma have found a decrease in the ciliary body compared to controls.[77]

No drug utilizing an endocannabinoid mechanism is currently available. Delivery mechanisms utilizing nanoparticle vehicles have been found to improve transcorneal penetration and AH dwell time.[78]

Conclusion and Future Studies

After contemplation of this review, one thing is clear: lipids play a significant role in IOP regulation.[79, 80] [Figure 3] The complete story of how is yet to be fully described. It is feasible that any one of the considered lipids could be a target of extensive study and a potential future pharmacological target. An area of particular interest would be to increase the AH outflow through the TM and SC. A rising quantity of studies are showing improvements in IOP in animal models treated with various lipid therapies, many of which target this very pathway. A particularly interesting realm in IOP research surrounds exosomes. The unique structure of the eye that results in a one-way flow of fluid (ciliary body to TM) in such a small system allows for powerful paracrine messaging. Exosomes appear to realize a role in the eye’s ability to cope and adjust to increased IOP. Lipids present a great challenge and opportunity for future investigation, most especially within the context of glaucoma medicine. Substantive research has demonstrated the promise of multiple lipid targets as novel therapeutic approaches to IOP regulation.

Figure 3:

General overview of the effects of lipid classes relevant to the aqueous humor and trabecular meshwork. Arachidonic acids (AAs) exert varied effects depending on their location in the anterior chamber and their product; derivatives such as prostaglandins and endocannabinoids are shown separately. Transcriptional control of lipid metabolism in the trabecular meshwork should also be considered when considering these relationships. Created using BioRender.com.

Highlights:

• Trabecular meshwork stiffness affects the aqueous humor outflow resistance and drainage.

• Lipids found in aqueous humor can alter the biomechanical properties of tissues in the trabecular meshwork outflow pathway.

• Lipid contents in the aqueous humor of glaucoma patients and controls differ significantly.

• The first-line treatment for glaucoma is prostaglandin drops, a lipid mediator derivative.

• Targeting de novo lipogenesis in the aqueous humor outflow pathway has shown promise in lowering intraocular pressure.