The Molecular Mechanisms of Trabecular Meshwork Damage in POAG and Treatment Advances

Abstract

Primary open-angle glaucoma (POAG) is the leading cause of irreversible blindness affecting over 60 million people worldwide. Elevated intraocular pressure (IOP) due to dysfunction of trabecular meshwork (TM) is the most significant and the only known modifiable risk factor for POAG. Although, glaucomatous TM damage is known to be mainly responsible for IOP elevation, none of the current treatments target TM pathology. This is partly due to an incomplete understanding of the pathophysiological mechanisms of TM damage. In this review, we summarized pathological changes of TM damage in POAG and our current knowledge of the mechanisms of glaucomatous TM damage, particularly focusing on linking the genetic factors of POAG (e.g., mutations and variants in POAG risk genes, risk loci, dysregulation of gene expression) to molecular pathways of pathogenesis in TM. In terms of treatment, reduction of IOP is the mainstream strategy that can be achieved by medical, laser or surgical treatment. IOP lowering drugs, laser or surgery can lower IOP, but do not reverse or restore the oxidative stress or other TM damage in POAG. Additionally, antioxidants, ginkgo biloba extract and nutrients could be a promising treatment for POAG.

1. Introduction

Glaucoma is a complex progressive neurodegenerative eye disease characterized by progressive loss of retinal ganglion cells (RGCs) axons [1]. It is the leading cause of irreversible blindness worldwide. Primary open-angle glaucoma (POAG) is the most prevalent form of glaucoma and is responsible for ~90% of all cases. POAG is a multi-tissue disease that targets, in ascending order, the TM, the optic nerve head, the lateral geniculate nuclei and the visual cortex [1]. As is known to us, TM dysfunction induces impaired aqueous humor (AH) drainage, elevated IOP and alterations of the optic nerve head and the visual field defects in POAG [2, 3, 4, 5].

As the drainage of the AH outflow pathway, TM cells (TMCs) are constantly exposed to different types of stress such as mechanical, oxidative, and phagocytic stress during their lifetime. TMCs have cellular defense mechanisms including the antioxidant system, proteolytic system, and regulation of stress-responsive genes that allow them to cope with daily challenges. Since TMCs are known to be highly differentiated cells characterized by a low renewal rate, injured cells are not readily replaced and damage is not diluted through cell division, leading to the progressive acceleration of TM damage resulting in glaucoma [6, 7].

Although, significant advances in ophthalmologic knowledge and practice have been made, the mechanisms responsible for TM damage are not yet completely understood. Current treatment for POAG revolves around controlling IOP by drug, laser or surgical treatment, rather than preventing, reducing or repairing TM damage. Therefore, up to now, there is no effective treatment able to ensure healing. In this review, we summarize our current knowledge of the pathological mechanisms of glaucomatous TM damage, particularly focusing on linking the genetic factors of POAG (e.g., mutations and variants in POAG risk genes, risk loci, dysregulation of gene expression) to molecular pathways of pathogenesis in TM.

2. Anatomical structure and biomarkers of TM and AH outflow pathway

The TM is a highly specialized tissue with a small size (100–150 μg, containing approximately 200,000–300,000 cells) located at the angle formed by the cornea and the iris in anterior chamber (AC) [8]. The TM consists of three regions: the uveal meshwork (UM) which is adjacent to the anterior chamber, corneoscleral meshwork (CM) which is located at the middle layer, and juxtacanalicular tissue (JCT) which is made up of TM cells embedded in the extracellular matrix. The JCT is adjacent to the inner wall of Schlemm’s canal (SC) and is considered to offer the major resistance to the AH outflow (Figure 1). The AH is generated in the ciliary processes from arterial blood. Then AH reaches the anterior chamber from the posterior chamber by passing the pupil and flows out through the TM. After crossing the TM, AH reaches Schlemm’s canal, which drains directly to the aqueous veins. The TM is the main pathway (called the conventional pathway) for modulating AH outflow resistance. Approximately 10% of the remaining AH leaves the anterior chamber through the uveoscleral pathway (unconventional pathway).

Figure 1.

The whole eye and the TM tissue were shown in both two-dimensionally and three-dimensionally to explain the AH outflow pathway. Normal IOP is required to maintain the proper physiological function and the structure of the globe of the eye. The IOP state critically depends on the balance between the inflow and outflow of AH. Malfunction of TM induces elevated IOP and alterations of the optic nerve head and the visual field defects. The TM consists of endothelial cells immersed in their fundamental substance. AH flows through the TM in both the intercellular route and the transcellular route.

Previously, AQP1, MGP, CHI3L1, TIMP3 and MYOC were used as typical TM markers. However, their application is hindered due to their limited specificity for distinguishing diverse cell types in a tissue. Larger-scale single-cell sequencing combining the in situ immunohistochemistry is a powerful strategy for revealing substantial cell markers for distinguishing different cell types in tissues. Recently, Patel et al. [9] and Zyl et al. [10] used single-cell RNA sequencing (scRNAseq) to identify the cell types in the human trabecular meshwork and the surrounding tissues, providing new insights into the cell types that comprise these pathways. Zyl et al. identified 19 cell types in these tissues with distinct molecular markers to define them using scRNAseq. The results of some key genes were validated in tissue by in situ hybridization or immunostaining. Among 19 cell types, 8 cell types (Clusters 3, 5, 8, 16, 15, 9,7, and 18) belong the conventional outflow pathway. Of them, cell types of Clusters 3, 5, and 8 are within the filtering TM region, with high expression levels of MYOC, MGP, PDPN, and RARRES1. Clusters 3 and 5 are distinguished by preferential expression of FABP4 and TMEFF2, respectively, each of which marked a subset of beam cells, Beam A and Beam B. Other clusters, such as Cluster 16, 15, 9, 7, and 18 correspond to cells adjacent to the filtering TM region with several new markers [10].

3. Pathological changes of TM damage in POAG

3.1 Abnormal accumulation of extracellular matrix (ECM) components in glaucomatous TM

In POAG, TMCs undergo a series of molecular and morphological alterations which lead to a gradual decrease in their cell number and IOP elevation. The increase of IOP, in turn, results in other pathological alterations that further impair cell homeostasis, leading to a vicious circle [11].

TM fibrosis is a key pathological characteristic of POAG [12, 13]. Fibrosis results in increased ECM deposits both in the TM and in the lamina cribrosa (LC). Both aberrant stiffness of TMCs and abnormal accumulation of ECM components contribute to TM fibrosis, leading to AH outflow resistance and elevated IOP [13]. During the process of POAG, the TM displays several alterations on morphologies and functions, including cell loss, increased heterogeneity of TM cellularity, increased accumulation of ECM, reduced adhesion of TMCs to ECM, formation of cross-linked actin networks, endothelial dysregulation, changes in the cytoskeleton, altered motility, reduced adhesion of TMCs to ECM, subclinical inflammation, progressive senescence and outflow impairment [11, 12, 13, 14, 15]. TM damage might trigger cross-linked proteins formation within aging tissues with malfunctioning proteolytic and ECM remodeling, as well as apoptosis and cell loss [13]. The AH proteome profile also undergoes dramatic changes, reflecting cellular and molecular damage to the TM [14, 16].

Transforming growth factor-β (TGF-β) signaling is widely recognized as a core pathway of fibrosis. TGF-β2 expression is increased in the AH and TM of POAG eyes [12]. Activation of TGF-β2 signaling causes a significant increase in oxidative stress in TMCs [17]. Several studies have demonstrated that TGF-β is involved in TM damage: (1) TGF-β induces mitochondrial ROS generation, (2) ROS are required for TGF-β induced gene expression downstream of Smad3 phosphorylation and nuclear translocation, (3) TGF-β induced transcription of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 requires mitochondrial ROS forming a feedback loop leading to increased intracellular ROS, and (4) blocking ROS generation markedly reduces TM profibrotic gene expression induced by TGF-β [18, 19].

Clinical evidence also suggests that POAG patients exhibit features of impaired NO signaling. NO is produced in vascular endothelia by the enzyme endothelial NO synthase (eNOS). The previous studies have confirmed that eNOS is an important protein in IOP regulation through the conventional outflow pathway. In the TMCs, NO has a critical role on the relaxation of TM [20]. The down-regulation of eNOS activity and reduced availability of NO is associated with POAG. In animal model studies, increased eNOS levels in the mouse TM have been associated with a reduction of IOP [21]. Stamer et al. [21] reported that mice overexpressing NOS3 had lower IOP and increased outflow facility than wild type in mice. Conversely, increased IOP and reduced outflow facility were observed in the NOS3 KO mice [22]. Furthermore, exogenous NO donor compounds can reduce IOP and increase outflow facility in several animal models [23]. Recently, Patel et al. [23] show that fluid flow-induced shear stress activates TRPV4TM channels and induces eNOS-mediated NO production. The activity of TRPV4TM channels is impaired in glaucoma that render TM cells are insensitive to fluid flow-induced shear stress. TRPV4 channel activator can lowers IOP and improve outflow facility by increasing eNOS activity and production of NO in TM cells. This indicates an important role for TRPV4-eNOS signaling in IOP regulation. In addition, studies have also demonstrated the association of polymorphisms in the NOS3 gene, which encodes eNOS, with the development of glaucoma [24].

4. Pathophysiological mechanisms of TM damage: implications for POAG pathogenesis

It is well known that several factors, including aging, genetic factors, environmental factors, and metabolites, involved in the onset and development of TM damage in POAG. Moreover, several mechanisms are triggered, leading to TM damage.

4.1 Oxidative stress

Oxidative stress is the exact and the most concerned mechanism leading to DNA, mitochondrial, and ECM damage in the TM and contributing to POAG pathogenesis [25, 26]. Several studies have demonstrated that TMCs are the most sensitive cell types to oxidant damage [25, 26]. The ability of TMCs to fight oxidative damage is critical to their survival and functions. Under normal circumstances, TM is exposed to a constant low level of oxidative stress and the expression of antioxidant enzymes makes TMC relatively resistant to oxidative damage. Excessive accumulation of reactive oxygen species (ROS) or an imbalance between oxidants and antioxidants may lead to oxidative stress in TMCs [27, 28]. The progress of POAG may be accompanied by a decrease in the antioxidant capacity of TMCs. Also, free radicals cause a gradual increase in oxidative damage, cytoskeletal changes and ECM accumulation in TMCs [29]. Oxidative damage to ECM adhesion results in damage to TM integrity, TM cell adhesion, and finally leads to cell loss [30]. As well, oxidative stress could damage the TMCs proliferation and migration function [17].

4.2 Inflammation

Inflammation is known to be increased in POAG patients [18, 19, 31]. The study revealed that cellular infiltration of immunocompetent cells (CD3+ and CD45+ cells) exists around the collector channels of the TM pathway in POAG [31]. Increased levels of inflammatory mediators, such as TGF-β1, TNF-α, and interleukins (e.g., IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-20 family) were found in the TMCs and the AH of POAG patients and animal models [31, 32, 33]. These inflammatory mediators can induce changes in the ECM and the TMCs cytoskeleton, and have been shown to be associated with the pathogenesis of POAG [33]. Some factors, such as IL-1α, which is highly expressed in TMCs, can up-regulate the expression of other inflammatory mediators [31, 32, 33]. The up-regulation of inflammatory mediators, e.g., IL-1α, and IL-1β, induced by oxidative stress in TMCs were reported [34]. In addition, both NF-κB and arachidonic acid are inflammatory components that are known to be activated in the TMCs to protect against oxidative stress in POAG eyes [35, 36]. Along with the continued activation of inflammatory responses, TM exhibits decreased cellularity and irreversible damage. Prostaglandin analogues converted from arachidonic acid are currently used for the treatment of POAG. These prostaglandin analogues reduce IOP by increasing the outflow of AH [37, 38]. These results reinforce the association between inflammation and POAG pathogenesis.

4.3 Vascular dysregulation and hypoxia

Primary vascular dysregulation (PVD) is another potential mechanism of POAG pathogenesis. Most POAG patients have signs of decreased ocular blood flow and ocular ischemia in the eye, indicating that hemodynamic factors are also involved in the POAG process [39, 40, 41]. In POAG eyes, oxygen tension in the tissue often falls temporarily and very mildly. This fall occurs repeatedly over years and leads to a preconditional adaptation, making the TMCs more resistant to coming oxygen fall. When the oxygen fall exceeds a certain critical value, reperfusion damage occurs. If the oxygen drop lasts longer or greater, it can cause tissue infarction. In POAG, reperfusion damage is very mild but recurrent as well [41]. Recurrently mild reperfusion induces chronic oxidative stress and inflammation, which harms a diversity of molecules and reduces cellular survival. IOP fluctuation is more damaging than a stably increased IOP [42]. All POAG patients with elevated IOP or normal IOP suffer from autoregulation disorders [43], for which the main cause is PVD contributing via ischemia, hypoxia and oxidative stress to TM damage. Meanwhile, systemic oxidative stress is also associated with decreased ocular blood flow [44]. In addition, nitric oxide (NO) is an endothelium-derived relaxing factor, which improves ocular blood flow [20]. In patients with glaucoma, decreased NO production has been found in AH of POAG patients compared with controls [45].

4.4 Endoplasmic reticulum (ER) stress

Recent studies have revealed the role of chronic ER dysfunction of TM in the POAG process [46]. The ER is a vast membranous network and interacts with the ribosome, Golgi bodies, proteasomes and mitochondria. ER is a central organelle for of synthesis, modification, folding and maturation of proteins. Accumulation of unfolded and misfolded proteins in the ER would trigger activation of the unfolded protein response (UPR) pathways [2, 47]. UPR could be activated by increased protein synthesis, inhibition of protein glycosylation, the presence of mutant or misfolded proteins, imbalance of ER calcium levels, energy deprivation, hypoxia, pathogens or pathogen and toxins. PERK, a type I ER transmembrane kinase, is activated by ER stress. The activation of PERK leads to phosphorylation of the eukaryotic translation initiation factor (eIF2α), resulting in translational repression. On the other hand, under ER stress condition, transcription factor 4 (ATF4) is upregulated that leading to the increase of C/EBP homologous protein (CHOP) [47]. The activation of PERK-ATF4-CHOP persists during chronic ER stress and triggers cell death [2, 47, 48]. Expression of either ATF4 or CHOP promotes aberrant TMCs protein synthesis and ER client protein load, leading to ECM accumulation and TM dysfunction. The damaged ER activates inflammation via NF-κB, mitochondrial damages, and enhanced TM cell apoptosis, which leads to elevated IOP [48].

4.5 Proteolytic system malfunction and compromised autophagy

The normal function of the proteolytic system in TM plays a role in preventing POAG. Misfolded and mutated newly synthesized proteins are rapidly degraded to prevent the toxicity caused by protein accumulation [49]. The major proteolytic system includes the ubiquitin-proteasome system (UPS) and autophagy. PERK, IRE1α and ATF6α that are the three ER stress sensors that can regulate UPS and autophagy during the ER stress [50]. The protein degradation process regulated by UPR has great significance for the maintenance of normal function of TM. In most cases, the degradation of excessive proteins protects TMCs with stress survival from apoptosis [51, 52].

Autophagy is a fundamental process for the degradation or recycling of intracellular components, which promotes cell survival or promotes cell death in an environment-dependent manner [49]. On one hand, basal autophagy under physiological conditions is a cellular self-protection mechanism, protecting cell survival in the absence of energy or nutrients and responding to cytotoxic insults, which is critical to maintain cell homeostasis in synthesis, degradation and recycling of cell compounds [49, 50]. On the other hand, excessive stress-induced autophagy may cause cellular stress in turn and promote apoptosis or autophagic cell death [50, 51, 52, 53]. Autophagy selectively eliminates unwanted, potentially harmful cytosolic material, such as damaged mitochondria or protein aggregates. It is apparent that autophagy is impaired in TM pathologies [50].

Acute stress can initiate autophagosome formations and autophagic degradation [54]. However, the TM pathological processes of POAG are long-term chronic procedures instead of acute changes. When TM cells are chronically exposed to oxidative or other stress, significant functional damage to their lysosome system has been observed. The accumulation of nondegradable ECM resulting from impaired autophagy accelerates cell senescence [55]. These harmful processes contribute to TM structural and functional alterations. Porter et al. [54] revealed the activation of autophagy responding to chronic oxidative stress in TMCs is mTOR-dependent.

4.6 Aging

Oxidant stress, inflammation, ischemia, hypoxia ER stress, protein aggregation, metabolic block and other stress events as well as impaired cellular repairability, have been found to be involved in the induction of senescence of TM [56]. Senescent TM tissue presents an alternated morphological and molecular phenotype, including TM becomes more pigmented, the scleral spur becomes more evident, the trabeculae become flatter and gradually merges into each other, and the occurrence of the denudation of trabecular areas [57]. The increase in thickness of the sheaths of the elastic-like fibers is also observed in the cribriform layer of TM during the aging. The molecular alterations in TM during the aging include increased anti-apoptotic gene expression, chronic activation of the DNA-damage response (DDR), increased activity of the β-galactosidase associated with senescence (SA-β-Gal), lipofuscin accumulations in lysosomes, lysosomes accumulations, increased number of defective mitochondria and the activation of unfolded protein response (UPR) in the endoplasmic reticulum (ER). Moreover, a reduced ATP release in response to mechanical stress and a severe dysregulation of calcium homeostasis which can contribute to TM age-related damage were observed in TM senescent cells [58]. Decreased cellularity is another character in aging TM tissue. An approximately 60% of reduction is observed in TM cellularity associated with aging from 0 to 80 years, indicating there are fewer TM cells in aged glaucomatous human eyes compared to young, healthy human tissue [58].

5. Abnormal intercellular communication

Extracellular vesicles (EVs) allow the exchange of nucleotides, proteins and lipids between cells and mediate intracellular communication, which may play a key role in TM function and the POAG pathogenesis [59]. EVs are important constituents of the AH, which participates in the communication between the non-pigmented ciliary epithelium (NPCE) and the TM [60]. AH carrying EVs is produced by the NPCE and flows into the posterior chamber, which then moves into the anterior chamber and is finally drained through the TM and into the venous system [61]. Intercellular communication can be achieved by EVs membrane proteins that interact with the TMCs through endocytosis, phagocytosis or act as ligands for cell surface receptors on TMCs [62, 63, 64]. Interestingly, when TM is exposed to EVs, its ability to resist oxidative stress is enhanced [65]. EVs-mediated cell-to-cell communications between NPCE and TMCs are involved in the IOP regulation. When TMCs receive the wrong signal carried by EV from NPCE, TMCs will not be able to respond sensitively to maintain IOP homeostasis. Recurrent incorrect response patterns may lead to TM dysfunction and morphologic alterations. The canonical Wnt signaling may involved in the regulation of IOP and in the effects of NPCE-derived EVs on TMCs [65].

6. Pathogenic genes associated with trabecular meshwork damage in POAG

A substantial fraction of glaucoma cases is influenced by genetic factors. About 5–10% of POAG is currently attributed to single-gene (e.g., MYOC, CYP1B1, GLIS3, LOXL1, LTBP2, PITX2, EFEMP1 and OPTN) or Mendelian forms of glaucoma [66, 67, 68, 69]. Many of the remaining cases of POAG may due to the combined effects of multiple genes and the interactions of gene-environment. Genome-wide association studies (GWAS) have demonstrated many genomic loci are associated with POAG risk, including CDKN2B-AS1, TMCO1, CAV1, CAV2, SIX1, SIX6, AFAP1, ABCA1, TXNRD2, FOXC1/GMDS, ATXN2, FNDC3B, ABO, PMM2, ATOH7, TMCO1, and GAS7 [69]. Recently, a large multi-ethnic meta-analysis of genome-wide association studies identified 127 POAG risk loci, and of which 44 loci were previously unreported [70]. These genetic risk factors affect the development of POAG through a variety of pathological processes (Figure 2). Here we focus on several genes involved in the maintenance of TM functions and pathological processes of POAG.

Figure 2.

Histopathological characters of trabecular meshwork (TM) damage in POAG. Implications for POAG pathogenesis: pathophysiological mechanisms of TM damage driven by oxidative stress and mitochondrial dysfunction, inflammation, vascular dysregulation and hypoxia, compromised autophagy, endoplasmic reticulum (ER) stress, abnormal intercellular communication and aging.

6.1 MYOC

MYOC is the first gene identified to be involved in POAG. MYOC encodes the secreted protein myocilin, which is highly expressed in the TM cells. Mutations in MYOC were found with a high prevalence rate in patients with POAG of various populations. However, studies demonstrate that overexpression or knockout of Myoc in mice does not cause glaucoma, hinting a gain-of-function mechanism may be involved [71, 72]. Accumulating evidences indicate that mutant Myoc is misfolded and accumulates within TM cells, which promote ER stress [73, 74]. The ER stress activates the UPR signal, which protects the TM cells, corrects misfolding, prevents translation of misfolded proteins, prevents translation of misfolded proteins and degrades misfolded proteins. However, excessive and sustained ER stress can trigger apoptosis in the TMCs, which then leads to an increase in resistance to AH outflow and elevated IOP, and, ultimately, glaucoma.

6.2 GLIS1 and GLIS3

GLI-similar 1-3 (GLIS1-3) constitute a subfamily of Krüppel-like zinc finger proteins that are involved in multiple biological processes by acting either as activators or repressors of gene transcription [75]. GLIS2 plays a critical role in the kidney and GLIS2 dysfunction leads to nephronophthisis, an end-stage, cystic renal disease [75]. GLIS3 plays a critical role in the regulation of multiple biological processes and is a key regulator of pancreatic β cell generation and maturation, insulin gene expression, thyroid hormone biosynthesis, spermatogenesis, and the maintenance of normal kidney functions [75]. GLIS3 genes have been associated with the increased risk of several diseases including glaucoma [75, 76]. GWAS studies have identified several SNP located in GLIS3, e.g., rs2224492 [76], rs736893 [77] and rs6476827 [78], associated with increased risk of POAG or raised IOP.

A previous study showed that GLIS1 significantly promotes the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) [75]. Recently, Gharahkhani et al. [70] conducted a large multi-ethnic meta-analysis of genome-wide association studies and identified more novel loci for POAG risk, including GLIS1 locus (rs941125). The study by Nair et al. [79] demonstrates that the mice lacking GLIS1 developed enlarged eyes and a long-lasting increase in IOP. The study revealed that low levels of GLIS1 induce the degeneration of the TM, leading to inefficient drainage of the AH in mice. In addition, they showed that GLIS1 regulates the expression of several glaucoma-associated genes, e.g., MYOC, LTBP2, LOXL1, TGFBR3, ADAMTS10, CYP1B1, EFEMP1, MMP2, and several ECM-related genes including collagen I, IV, ADAMTS10, FBN2, LOXL1–4, and VI families, LTBP2, a regulator of TGFβ signaling and ECM deposition. In addition, the researchers also identified rs941125 at GLIS1 gene in humans are linked to risk of POAG. All these results indicate that GLIS1 is a key regulator in TMCs and a risk gene of glaucoma.

6.3 FOXC1

FOXC1 belongs to the Forkhead box (FOX) family of transcription factors and it is expressed in the adult eye including the TM. FOXC1 has been identified as a susceptibility locus for POAG and elevated IOP in several GWAS studies [76, 78, 80]. Mutations in the FOXC1 gene can cause Axenfeld-Rieger syndrome (ARS), a developmental disorder affecting structures in the anterior segment of the eye with an increase in IOP [80]. More than half of ARS patients with FOXC1 mutations will develop earlier-onset glaucoma. FOXC1 is expressed in ocular tissues including TM. Several genes that have TM relevant functions including miR-204, OLFM3, CXCL6, MEIS2, LDLRAD2, CLOCK and ITGb1 expressions was affected by FOXC1 in TM cells [81]. FOXC1 has been demonstrated to be a critical regulator for stress response [82]. The TM is the most sensitive tissue of the anterior segment of the eye to oxidative stress. Studies demonstrate that reduced FOXC1 expression increases cell death in cultured TM cells in response to oxidative stress, suggesting FOXC1 have a role in preventing cell death under both normal and oxidative stress conditions [82, 83]. HSPA6, a member of the heat-shock 70 family of proteins, has been identified as a target gene of FOXC1 [83]. HSPA6 protein is induced under severe oxidative stress conditions, has a protective function in TM cells. A decrease in FOXC1 results in the repression of several anti-apoptotic genes, including FOXO1A [82]. FOXO1A is a key protein in cellular stress response and apoptotic pathways, its expression is directly regulated by FOXC1 in TM cells [82]. Studies have shown that the FOXC1 gene also was involved in the regulation of prostaglandin receptor genes [84]. Doucette et al. [84] confirmed that FOXC1 binds to enhancer element of EP3 gene prostaglandin receptor (PTGER3) and activates PTGER3 expression. Other prostaglandin receptor genes EP1 (PTGER1), EP2 (PTGER2), EP4 (PTGER4), and FP (PTGFR) were altered when FOXC1 was knocked down in culture TM cells. This study provides a clue to explain why some glaucomatous patients do not respond to treatment of Prostaglandin analogs. Prostaglandin analogs (e.g., latanoprost, bimatoprost, travoprost, and tafluprost) are the frontline medications used to lower IOP in glaucomatous patients. However, about 30% of patients do not respond to these medications. Furthermore, 50–60% of patients with secondary glaucoma caused by FOXC1-mediated ARS did not respond to these medications. FOXC1 mutation or reduction of expression leads to the dysregulation of the prostaglandin signaling pathway, which probably account for the lacking of response to prostaglandin-based medications [84].

6.4 ATXN2

ATXN2 is a ubiquitous RNA-binding protein with a polyglutamine (polyQ) CAG repeat in its coding region. ATXN2 has roles in regulating many cellular processes, including stress granule formation, starvation and stress response, translation, RNA processing, metabolism, mitochondrial function, and calcium signaling [85, 86]. Expansions of polyglutamine repeat of ATXN2 have been implicated in spinocerebellar ataxia 2 (SCA2) and amyotrophic lateral sclerosis (ALS) [83]. In addition to causing SCA2 and increasing the risk of developing ALS, mutations in ATXN2 may play a role in a handful of other diseases, including Parkinson’s disease (PD), spinocerebellar ataxia type I (SCA1), Machado-Joseph Disease (SCA3), tauopathies, POAG, obesity and type I diabetes [85, 86].

In eukaryotic cells, various cellular stresses (e.g., starvation, heat shock, ER stress, oxidative stress) elicit the formation of cytoplasmic stress granules (SGs) as a part of the homeostatic response [87]. SGs contain non-translating mRNAs, translation initiation factors, and RBPs, which protect cells from damaging signals and suppress general translation. SG formation is beneficial for cell survival by preventing the accumulation of misfolded proteins and mutant proteins. Furthermore, upon the environmental stresses, SGs sequester several apoptosis regulatory factors into granules and thereby inhibit stress-induced cell death signaling. Since TM cells are exposed to various stresses simultaneously during their lifetime, the formation of SGs under multiple stress conditions is important for the function maintenance of TM cells.

Mutations of ATXN2 have been linked to impaired formation of stress granule in normotensive glaucoma, primary open-angle glaucoma, SCA2, and ALS [85]. ATXN2, ATXN2L, and their associating proteins have been identified as key components of mammalian SGs. Depletion of ATXN2 suppresses the SGs formation. ATXN2 repeat expansions impair the assembly of stress granule, leading to stress-granule-induced cytotoxicity and neurodegeneration. Studies show that inhibition of SGs assembly could promote apoptosis of cells [88]. Clearing of SGs involves in the autophagy pathway. Polyglutamine expansions in ATXN2 have been associated with autophagic and mitochondrial dysfunction in several neurodegenerative diseases. The study finds that the SCA2 cells expressing expanded ataxin-2 are particularly susceptible to autophagic inhibition when cells were treated with autophagy inhibitor chloroquine [89]. This treatment led to more apoptosis in SCA2 cells compare to controls, hinting that the SCA2 cells are more susceptible for autophagic inhibition [89]. This could be explained that ATXN2 is an inhibitor of mTOR signaling [90]. MTOR has been characterized as a negative regulator of autophagy [91]. This suggests that mutation of ATXN2 may lead to compromised autophagy by reducing inhibition of mTOR signaling.

Besides, GWAS study have identified ATXN2 were associated with risk for POAG [78, 79]. Expression analysis reveals that ATXN2 is expressed in the cornea, trabecular meshwork, ciliary body, retina and optic nerve [92]. It has the strongest expression in RGCs [92] and this result is confirmed by the expression profile from scRNAseq [10]. The expression of ATXN2 in key POAG-relevant ocular tissues supports its potential role in autophagy and stress granule formation in response to ocular hypertension.

6.5 EFEMP1

EFEMP1 encodes fibulin-3, an extracellular matrix protein that serves to modulate cellular behavior and functions by connecting and integrating multiple partner molecules in the extracellular compartment. It is expressed in the retina and RPE, involved in age-related macular degeneration (AMD) [93, 94]. A GWAS identified copy number variation (CNV) at NPHP1 and EFEMP1 as potential candidates for the association of inherited retinal degenerative diseases [95]. The EFEMP1 gene was located within the GLC1H locus on chromosome 2p (2p15-p16). A report located a genetic locus (GLC1H) for adult-onset POAG maps to the 2p15-p16 region with linkage analysis in an Afro-Caribbean (Jamaican) population [96]. In another study, a region on chr2 (chr2: 46.4 M–65.6 M) was located that contributed to POAG with family-linkage analysis studies in Chinese [97, 98], which is overlapped with the 2p15-p16 region. A study identified a novel missense variant (p.Arg140Trp) in exon 5 of the gene coding for EFEMP1 that cosegregated with POAG in an African-American family [99]. Recently several GWAS studies have demonstrated that EFEMP1 is associated with increased risk for increased IOP for POAG [66, 78, 100]. In addition, mutations in EFEMP1 also were identified in sporadic POAG patients [68]. EFEMP1 is regulated by GLIS1 in TM cells [79]. Altered cell-ECM interaction or abnormal ECM organization was observed in Glis1-KO mice [79]. Furthermore, analysis of single-cell sequencing revealed that EFEMP1 is high expressed in Fibroblast, JCT and BeamB in TM region [10], indicating EFEMP1 plays a critical regulatory role in maintaining the structural integrity and functionality of the TM. As described in the previous section, TGF-β2 is significantly increased in the AH of patients with POAG. Expression analysis showed that EFEMP1 can be downregulated by TGF-β2 in cultured HTM cells [101], indicating the function of EFEMP1 may be impaired in TM in some POAG patients.

6.6 LOXL1

LOXL1 is a member of the lysyl oxidase family involving in extracellular matrix formation. This enzyme is required for linking collagen and elastin in connective tissues and catalyzing the polymerization of tropoelastin to form the mature elastin polymer. Previously, it was thought that the LOXL1 gene was associated only with XFG [102]. The allele T for the intronic SNP (rs2165241), and the allele G for both coding SNPs (rs1048661 and rs3825942) are associated with a higher risk of XFS and XFG in the studied population [102]. It seems that low levels of LOXL1 expression could predispose to XFS. No association was seen with POAG in that study. However, several recent GWAS studies in different ethnic populations have demonstrated that LOXL1 was associated with risk for POAG [70, 103, 104, 105].

LOXL1 expression is detected in ocular tissues such as lamina cribrosa, lens epithelium, cornea, ciliary muscle, and trabecular meshwork, all of which are mainly involved in the formation of the extracellular matrix. Profile of expression from scRNAseq revealed LOXL1 is predominantly expressed in TM (BeamB, JCT, and BeamA) [10]. In TM cells, LOXL1 is regulated by GLIS1. When GLIS1 was knocked down by shRNA lentivirus in HTM cells, the expression of LOXL1 was reduced [102]. ChIP-Seq analyses revealed that LOXL1 is directly regulated by GLIS1 protein binding to its promoter [79].

Glaucoma is age-related disease in human. Recent research suggests that epigenetics, especially DNA methylation, plays a critical role in aging. One proposed cause of aging is the disruption of epigenetic-sensitive molecular networks, which lead to decreased tissue function. Several evidences of LOXL1 epigenetic silencing by promoter methylation were reported in cancer and Cutis Laxa, a disorder of connective tissue [106, 107, 108]. Ye et al. reported that the promoter region of the LOXL1 gene was hypermethylated in patients with Pseudoexfoliation Glaucoma (PXFG) compared with controls, leading to a reduced expression of its protein product and downstream impaired elastic fiber homeostasis [107]. Similarly, Greene et al. [109] also discovered that the LOXL1 promoter methylation was increased in patients with PXFG compared to Control. These results indicated that hypermethylation of CpG islands in the LOXL1 gene may function as an essential mechanism in the pathogenesis of PXFG Glaucoma. In addition, the rare variant that probably impacts the function of LOXL1 protein was discovered in sporadic cases of POAG [68]. In our recent report, we found a rare variant (p.Cys448Phe) occurred in the LOXL1 protein lysyl oxidase domain [68]. This site is a conserved residue since the 443–456 sites are highly conserved in this domain, and several copper ion binding sites (His449, His451, and H453) are located in this region. It is worth noting that Cys448 and Cys497 will form a disulfide bond and the alternation of amino acid (from Cys to Phe) may lead to breakage of the S–S bond. These multiple lines of evidence indicate that the loss or decrease of LOXL1 function is related to Glaucoma, and it may also be an important risk factor for POAG.

7. Treatment

Different worldwide treatment recommendations and guidelines exist for the management of POAG [110, 111, 112, 113]. IOP is the main modifiable risk factor proven to alter the disease course in these guidelines. IOP lowering can be achieved by medication, laser or surgery (either alone or in combination). Moreover, nutrients in the foods that we consume every day can alter gene expression in cells, thereby exerting a beneficial or harmful physiological effect. Lifestyle, exercise, and nutrients therefore play a key role in eye health and could be used as an adjuvant in POAG therapy [114, 115].

Re-cellularization of the trabecular meshwork (TM) using stem cells is a potential novel treatment for POAG. Recent experimental studies demonstrated the potential effectiveness of regenerative therapies using iPSCs or TM progenitor cells in restoring TM tissue and reducing IOP. however, the potential plasticity and the lack of definitive cell markers for TM cells compound the biological challenge. Morphological and differential gene expression of TM cells located within different regions made it difficult to regenerate [116]. Here, we will not describe the detail of these novel therapies.

7.1 IOP-lowering drugs

Topical IOP-lowering medications have long been the POAG treatment and are widely used. The prostaglandin analogues reduce IOP by increasing the outflow of AH, primarily through the uveoscleral pathway [37]. They have also been shown to remodel ECM within the TM and reduce outflow resistance [38]. Prostaglandins became the first-line medication for POAG because of their IOP-lowering efficacy, once daily application and minimal systemic side-effects. Long-term use of PGA has been reported to decrease the central corneal thickness due to activation of corneal stromal matrix metalloproteinases (MMPs) [117, 118, 119, 120]. The other ophthalmic medication classes used in clinical practice include the beta-adrenergic blocking agents, the alpha-2 adrenergic agonists, and the carbonic anhydrase inhibitors [118, 119]. They can cause multiple ocular and systemic side-effects with poor compliance, which limits their clinical use.

Newer medical treatments are in development, including trabodenoson (a highly selective adenosine-1 receptor agonist), netarsudil (a Rho-kinase inhibitor and norepinephrine transporter inhibitor), latanoprostene bunod (a modified prostaglandin analogue), ONO-9054 (a novel non-selective prostanoid receptor agonist, dual EP3 and FP agonist) and others. Theoretically, most of these medicines are IOP-lowering treatments with new mechanisms of action, better efficacy, tolerability and convenience. The results of clinical trials including phase 3 trials were concluded successively [120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130].

As novel candidates for POAG, the ATP channel openers have been reported to protect the retinal ganglion cells during ischemic stress and glutamate-induced toxicity suggesting a neuroprotective property for this drug class [131].

7.2 Lasers

Selective laser trabeculoplasty (SLT) is a viable alternative ophthalmic treatment when patients experience ocular or systemic side effects due to medication. SLT is relatively safe and well-tolerated with low complication rates [132]. Recently, the laser in glaucoma and ocular hypertension (LiGHT) study [133] has evaluated treatment-naive patients with POAG or ocular hypertension randomly allocated to receive either initial primary SLT or initial topical medication. The LiGHT study demonstrates that there is no difference in health-related quality of life (HRQL) between primary SLT and initial topical medication at 36 months. It provides good IOP control, at a lower cost and allowed almost 74% of patients to be successfully controlled without drops for at least 3 years after starting treatment. The study demonstrates that SLT is safe and effective as a first-line treatment for POAG and should be offered as an alternative to IOP-lowering topical medication. Other laser trabeculoplasty procedures include Micropulse Diode Laser Trabeculoplasty (MDMT), Titanium Sapphire Laser Trabeculoplasty (TLT) and Pattern Scanning Laser Trabeculoplasty (PSLT) [132, 133]. Some small studies have compared their efficacy against SLT and found their potential advantages. However, larger studies are required to support whether any of them provide extra advantages over existing SLT.

7.3 Surgery

Several different glaucoma IOP-lowering surgeries exist, including penetrating (trabeculectomy, tube surgery, etc.), and non-penetrating surgery (deep sclerectomy, visco-canalostomy, and canaloplasty, etc.). Their use in clinical practice should consider not only the evidence of lowering intraocular pressure and the safety of each operation, but also the individual patient’s status. The decreases of IOP often were observed after cataract surgery [134, 135]. Cataract surgery has been described as the single best glaucoma surgery due to its IOP-lowering effect. Thus, clinicians may serve phacoemulsification as a valid treatment option for newly diagnosed POAG. The incisional operation generally achieves greater IOP lowering compared to medication and is usually performed if IOP lowering is insufficient by medication or laser [135]. But it may be a viable first option in those newly diagnosed POAG patients with a poor compliance or intolerant to medication. The Treatment of Advanced Glaucoma Study (TAGS) is currently investigating whether the surgical intervention could be the first treatment option in newly diagnosed advanced POAG [136].

7.4 Ginkgo biloba extract

G. biloba has been used as traditional herbal medicine for hundreds of years in China [137]. G. biloba extract (GBE) contains two groups of active substances: flavonoid glycosides including quercetin, rutin, and terpene lactones including ginkgolides A, B, C, and bilobalide [137, 138]. All these substances are now widely used as dietary supplements for decades in the world [137]. It has been used as a brain tonic to enhance memory, to decrease mental fatigue, and to improve concentration. Additionally, it is used to treat vertigo, tinnitus and vitiligo, to improve visual and auditory acuity, and in various neurological and psychological disorders such as dementia, cognitive decline and functional disability [137]. GBE has also beneficial effects on the cardiovascular system and has been claimed to prevent atherosclerosis [139]. Currently, its application in glaucoma is garnering much attention [140]. Flavonoids that are often found in G. biloba could increase ocular blood flow and potentially delay the progression of vision loss [141]. Several properties of GBE can be useful in the treatment of POAG by protecting TM and other non-IOP risk factors. GBE has been shown to act as an antioxidant and free radical scavenger, a membrane stabilizer, an inhibitor of the platelet-activating factor, a vasodilator, and a regulator of metabolism [142]. Several experimental studies have demonstrated the efficacy of GBE in reducing free-radical damage and lipid peroxidation and protecting the vascular endothelium [143, 144]. GBE may increase microvascular blood flow of various tissues including the eyes, brain and others, perhaps by reducing the viscosity of the blood. GBE has been found to increase perfusion in skin and nail bed capillaries without changing laboratory coagulation parameters [38]. Indeed, a study showed that GBE could improve ocular blood flow, without affecting blood pressure, heart rate or IOP [145]. Another study demonstrated that GBE significantly suppressed steroid-induced IOP elevation and prevent TMCs from damage in animal model [146]. GBE was also proved to protect the retina and macule from degeneration as well [147]. We speculate that there might be some synergy effects between GBE and drugs or other support substances. It seems that GBE increases their concentration in ocular through increasing ocular blood flow, that seems to be a “targeted drug delivery”.

7.5 Nutrition with antioxidative properties

Antioxidants can be grouped into two categories: enzymatic and nonenzymatic [148, 149, 150]. The enzymatic antioxidants, including superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione-S-transferase to scavenge the ROS in the body.

7.5.1 The enzymatic antioxidants

Disorders of glutathione peroxidase (Gpx) and nitric oxide synthase (NOS) are associated with glaucoma. Gpx can regulate ROS, while the NOS can produce nitrogen species (RNS). Lower plasma levels of both glutathione and glutathione peroxidase were found in POAG patients compared to controls [151]. In contrast, a significant increase in glutathione peroxidase activity in the AH and plasma of POAG patients have been reported by others [148, 149]. This divergence might be caused by the fact that antioxidant defense is decreased in patients with glaucoma, which results in an increase in the activity of antioxidant enzymes in an attempt to counteract the damage caused by ROS.

Ingested nitrate is turned into the vasodilator NO which improves ocular blood flow [148, 149, 150]. NO is also produced by the endothelium of the TM. The abnormal function of TMCs is associated with reduced NO bioavailability. The TM experiences physiological shear stress which triggers NO production. These cells may get lost as glaucoma progresses.

7.5.2 The nonenzymatic antioxidants

Non-enzymatic antioxidants including vitamins, carotenoids, polyphenols and flavonoids were intensively studied in POAG pathology [148]. Intake of vitamin A, C, E, B3 (nicotinamide), showed a beneficial association with glaucoma, improve inner retinal function [152, 153, 154]. Low vitamin D has been identified by some studies as an independent risk factor for glaucoma [155]. Vitamin D probably serves as an anti-inflammatory agent in the oxidative stress-driven pathogenesis of POAG. Coenzyme Q acts as another antioxidant similar to vitamins and was suggested to have the neuroprotective efficacy in glaucoma models [156]. In clinical studies, increased levels of carotenoids in macular pigment can help improve visual performance in glaucoma patients [157]. Astaxanthin features some important biologic properties, mostly represented by the strong antioxidant, anti-inflammatory and antiapoptotic activities. Both astaxanthin and saffron might be efficacious in the prevention and treatment of glaucoma [158, 159].

Polyphenols are plant-derived organic substances. Polyphenols can be divided into different subclasses, according to the number of phenolic rings present in their structure [160]. They comprise 4 families: simple phenolic acids, stilbenes (e.g., resveratrol), coumarins and flavonoids. Flavonoids include anthoxanthins, flavanones, flavanonols, flavans and anthocyanins. Flavonoids are widespread in nature, being found in a vast range of plants, including citrus fruits, grapes, tomatoes, berries and green tea; more than 5000 compounds that exert beneficial effects on health are known. These substances can protect cells or mitochondria from oxidative stress through different mechanisms and could offer therapeutic benefits to POAG patients [160].

Quercetin is a natural flavanol antioxidant and it has been reported to have notable curative effects on the treatment of glaucoma [161]. Baicalein has antioxidant and anti-inflammatory properties and can improve the treatment of glaucoma [162]. Curcumin reduces the inflammatory response by inhibiting the release of TNF-α and C-reactive protein and induces the expression of antioxidant enzymes or cytoprotective proteins [163, 164]. Resveratrol has been shown to increase the survival of retinal ganglion cells following ischemia-reperfusion injury for glaucoma in a study [165]. Meanwhile, resveratrol protects optic nerve head astrocytes from oxidative stress-Induced cell death through inhibiting the activation of caspase-3 activation, the dephosphorylation Ser 422 of Tau and the formation of misfolded protein aggregates [166]. Hesperidin, betalain and trehalose exert protective effects against glaucoma [167, 168, 169]. Polyphenols reduce inflammation through several mechanisms, such as reducing the expression of cytokines like IL-2, IL-6 and TNF-alpha [160]. Salidroside could inhibit TGF-β2-induced ECM expression in TMCs, and lower IOP which was elevated by TGF-β2 overexpression in mouse model [170]. Myricetin, is present in apples, oranges, berries, and vegetables. As a flavonoid, it can reduce oxidative stress and improve ocular blood flow in POAG. In POAG TMC, myricetin can substantially down-regulate the expression of TGFβ1/β2 [171, 172]. Myricetin effectively prevented IOP elevation and decreased IL-1α, IL-1β, IL-6, Il-8 and TNF-α) in the AH and TMCs in glaucoma rat model [172]. The results of these suggest that the intake of antioxidants in the diet could reduce the risk of glaucoma. The evidence is not conclusive; thus, more researches and long-term observations are required to evaluate the role of nutritional supplementation in glaucoma. The systemic status of these antioxidants in the tear, aqueous and vitreous fluid, as well as plasma, is a prospective gap in research.

7.5.3 Omega 3 fatty acids

Omega-3 (ω-3) fatty acids belong to the long-chain polyunsaturated fatty acid (PUFA) family. They have several properties that make them a potential adjuvant therapeutic agent in POAG. The first is that derivatives of ω-3 are the eicosanoids. These include the prostaglandin analogues, which are known for their IOP-lowering effect [38, 173]. Meanwhile, ω-3 exerts a highly protective effect on endothelial cells [174]. Omega-3 PUFA could reduce blood viscosity, probably because they improve the deformability of the red blood cells [175]. The anti-inflammatory properties of Omega-3 have also been demonstrated, which may have therapeutic potential for chronic inflammatory diseases such as glaucoma [176]. A high omega-3:6 ratio is recommended. Wang et al. [177] found that increasing the daily dietary intake of PUFA, including ω-3 fatty acids, was associated with a significant decrease in the probability of POAG. Finally, oral omega-3 supplementation for 3 months has been seen to significantly reduce IOP in normotensive adults [178] and in pseudoexfoliative glaucoma [179].

8. Conclusion

More and more evidences show that the onset of glaucoma is a result of the interaction of age, external environmental factors and genetic factors. The external environmental factors gradually impair the function of some key genes in the TM cell through a variety of ways, including decreased gene expression by cytokines, or epigenetic modification, which gradually change the phenotype of cells. Similarly, genetic factors (e.g., mutations of gene or polymorphism of POAG-associated genes) also lead to the gradual function impair in TM cells by influencing key regulatory pathways. There are some common signal pathways affected by genetic factors and environmental factors. Thus, the detailed characterization of the molecular profile of pathological and normal TMCs is critical to discover key regulatory molecules and pathways, which is the foundation for discovering the potential therapeutic targets. Simply reducing IOP by drugs, laser or surgery is not sufficient to guarantee a good prognosis in this disease. Therapies that focus on restoring TM cellularity and function could offer therapeutic benefits to POAG patients. The search for bioactive compounds with a protective effect on TM is of particular interest.

Acknowledgments

This work was supported in part by the projects (cstc2020jxjl130015, cstc2021jcyj-msxmX0767, and cstc2018jcyjAX0460) from the Natural Science Foundation of Chongqing.