Role of MicroRNAs in the Trabecular Meshwork

Abstract

MicroRNAs (miRNAs) are now recognized as important post-transcriptional regulators of gene expression. MiRNAs are known to modulate cellular functions relevant to the normal and pathological physiology of the trabecular meshwork (TM) such as cell contraction and extracellular matrix turnover. There is also increasing evidence supporting the role of miRNAs in the pathogenesis of multiple diseases, and their potential value as both biomarkers of disease and therapeutic targets. However, compared with other tissues, our current knowledge regarding the roles played by miRNAs in the TM is still very limited. Here, we review the information currently available about miRNAs in the TM and discuss the main challenges and opportunities to incorporate the rapid progress in miRNA biology to the understanding of the normal and pathological physiology of the TM, and to develop novel clinical applications for diagnosis and therapy of high intraocular pressure.

Introduction

MicroRNAs (miRNAs) are a class of small non-coding RNAs (19–25-nucleotides) found in plants and animals, that negative regulate gene expression.¹ MiRNA genes can be either intergenic or intragenic. Intergenic miRNAs have been found in the introns of protein and non-protein coding genes and in exons of long non-protein coding transcripts and their expression is usually regulated together with that of their host genes.^2–4 Intergenic miRNAs are believed to be transcribe independently from other genes and can be located in clusters that generate polycistronic units containing multiple discrete loops from which mature miRNAs are processed.^5,6 The promoters that regulate the expression of these miRNAs have been shown to have some similarities in their motifs to promoters of other genes transcribed by RNA polymerase II such as protein coding genes.^5,7,8 MiRNAs are transcribed as pri-miRNAs that are converted in the nucleus into 70-nucleotide stem-loop structures known as precursor microRNA (pre-miRNAs). These pre-miRNAs are then transported to the cytoplasm where they are processed by the endonuclease Dicer to mature miRNAs, 19–25 nucleotides in length. Mature miRNAs integrate into an RNA-induced silencing complex (RISC) and provide the specificity for binding to target sites of the specific mRNA transcripts. Animal miRNAs typically exhibit only partial complementarity to their mRNA targets and the specificity of the miRNA/mRNA interaction appears to be highly dependent on a “seed region” of about 6–8 nucleotides in length at the 5′ end of the miRNA.⁹ Binding of active RISC to a transcript can inhibit translation or induce mRNA degradation by argonaute proteins, the catalytically active members of the RISC.¹⁰

Although an miRNA-like mechanism for repression of gene expression was postulated in 1961 by Jacob and Monod,¹¹ the first miRNA was discovered in Caenorhabditis elegans by Victor Ambros' laboratory in 1993.¹² At the same time Gary Ruvkun's laboratory identified the first miRNA target gene.¹³ Only after 2000 additional miRNAs were characterized in multiple species demonstrating that miRNAs were not a C. elegans idiosyncrasy, but an important mechanism of gene expression regulation present in many organisms.^14,15

MiRNAs are well conserved in eukaryotic organisms¹⁶ and the increase in the number of miRNAs in the metazoan lineage has been shown to be associated with major body-plan innovations and the emergence of phenotypic variation in closely related species.^9,17 Further, analysis of the 3′-untranslated regions (UTRs) of orthologous neuronal genes showed that the density of miRNA target sites in invertebrates has increased in parallel with the complexity of the central nervous system.¹⁸ These observations suggest that the evolutionary expansion of miRNA families and target sites, rather than genome duplication events, might be a causal factor in the observed increase in complexity observed in metazoan evolution.¹⁹

Currently, the human genome is believed to encode over 1,000 miRNAs²⁰ that are abundantly expressed in many cell types and are estimated to target at least 60% of mammalian genes.^21,22 Under a standard nomenclature system, miRNAs are named using the “miR” prefix and a unique identifying number that often indicates the order of discovery (eg, miR-146). The names for lin-4 and the let-7 are the only exceptions to this rule, and they are retained for historical reasons. The uncapitalized prefix “mir-” refers to the pre-miRNA, while a capitalized “miR-” refers to the mature form. MiRNAs with nearly identical sequences except for 1 or 2 nucleotides have the same number followed by a lower case letter (eg, miR-146a and miR-146b). miRNAs for which the mature sequences are 100% identical but are located at different places in the genome are indicated with an additional dash-number suffix (eg, miR-146a-1 and miR-146a-2). The species of origin is designated with a 3-letter prefix such as “hsa” for Homo sapiens and “mmu” for Mus musculus (eg, hsa-miR-146a-1 and mmu-miR-146a-1). When 2 mature miRNAs originate from opposite arms of the same pre-miRNA, they are denoted with a -3p or -5p suffix (eg, hsa-146a-3p and hsa-146a-5p). An asterisk following the name can be used to indicate that an miRNA is expressed at low levels relative to the miRNA in the opposite arm of a hairpin (eg, hsa-miR-146a-3p is referred to as hsa-miR-146a* since its expression is lower than that of hsa-miR-146a-5p, which is frequently named hsa-miR-146a).^23,24

Much of the current research on miRNAs is focused on the elucidation of miRNA on cell pathology and physiology, typically using the gene expression profiling approach. Elucidating miRNA function is particularly complex because each miRNA can control translation of tens or even hundreds of different genes, and a single messenger can be targeted by multiple miRNAs. However, the capacity of miRNAs to simultaneously modulate entire networks of genes in a coordinated manner also makes miRNAs particularly powerful tools to effectively modify cellular behavior for experimental and therapeutic purposes.

MiRNAs have already been shown to participate in the regulation of most cellular functions,²⁵ and there is also substantial evidence implicating miRNA expression changes on multiple diseases.^26–29 The clinical relevance of genetic mutations affecting miRNA genes or miRNA binding sites has not been thoroughly investigated. However, some functional polymorphysms in miRNA genes have been discovered that might be relevant in the progression of diseases such as acute lymphoblastic leukemia³⁰ and diabetic retinopathy.³¹ Over the past few years, several examples of these functional miRNA binding site single nucleotide polymorphisms have been identified as biomarkers for different pathologies including cancer³² and rheumatoid arthritis.³³

An additional area of interest in miRNA research is the use of circulating miRNAs as biomarkers of human diseases. Multiple cell types have been shown to release miRNAs into the circulation and the miRNAs profiles in the plasma and/or serum are altered in several pathologies and over 100 circulating miRNAs have been identified as biomarkers for different diseases.^34,35 Extracellular miRNAs are known to be packaged by at least 3 methods: in lipid vesicles, such as exosomes, microvesicles, and apoptotic bodies; bound by RNA-binding proteins, such as nucleophosmin 1 and Argonaute 2; and associated with high-density lipoproteins.^36–39 These packaging mechanisms make miRNAs present in serum and other fluids resistant to circulating ribonucleases and severe physicochemical conditions.⁴⁰ Changes in abundance of circulating miRNAs have already shown some promise as diagnostic and prognostic markers in a variety of pathological conditions such as hypertrophic cardiomyopathy,⁴¹ cancer,⁴² myocardial infarction,⁴³ and hematological diseases.⁴⁴ Changes in extracellular miRNAs present in the vitreous humor show differences also in some ocular diseases.⁴⁵ However, glaucoma, so far, is not one of them.

Finally, because of the ability of miRNAs to modify cellular functions and their involvement in multiple pathologies, there is a great deal of interest in developing new technologies for their therapeutic use including ocular diseases such as age-related macular degeneration.⁴⁶ Several miRNAs have progressed in pre-clinical trials as therapeutic targets.⁴⁷ Phase 2 clinical trials are being conducted for treatment of hepatitis C virus (HCV) infection liver-expressed miRNA-122 using the locked nucleic acid (LNA)-modified antisense oligonucleotide miravirsen,⁴⁸ and phase 1 clinical trials have been initiated in patients with unresectable primary liver cancer or metastatic cancer with liver involvement by Mirna Therapeutics (Austin, TX).

Compared to other tissues, our current knowledge about the role of miRNAs in the trabecular meshwork (TM) is still very limited. Here, we present a review of the current knowledge about miRNAs in the TM and discuss the main challenges and opportunities to incorporate the rapid progress in miRNA biology to the understanding of the normal and pathologic physiology of the TM and to develop novel clinical applications for diagnostics and therapy of glaucoma. We will start reviewing the current information available about expression of miRNAs in human TM (HTM) cells and their alterations associated with cellular senescence and exposure to cyclic mechanical stress. Then, we will present current data supporting the role of miRNAs in the regulation of cellular functions relevant to the physiology of the outflow pathway such as cellular contraction and extracellular matrix (ECM) dynamics. Finally, we will discuss specific areas of interest for future research on miRNAs in the TM.

Changes in miRNA Expression Associated with Cellular Senescence in HTM Cells

Increased accumulation of senescent cells in some tissues has been implicated in aging and age-related pathologies.^49–54 Some experimental evidence suggests that the number of senescent cells in the TM increases with aging and in primary open angle glaucoma (POAG).⁵⁵ In addition, senescent cells present phenotypic changes that could contribute to the malfunction of the outflow pathway in glaucoma.^56–59 For this reason, the first analyses of miRNA expression in HTM cells were aimed at evaluating the potential role of miRNAs in shaping the phenotypic changes associated with the induction of cellular senescence as a result of chronic exposure to oxidative stress [stress induced premature senescence (SIPS)] or the exhaustion of the replicative potential of the cells (replicative senescence). These studies helped to identify consistent and significant changes in expression of 25 miRNAs associated with SIPS and 18 with replicative senescence.^60,61

Consistent with an antioncogenic role of cellular senescence, both SIPS and replicative senescence of HTM cells lead to a significant downregulation of several known oncogenic miRNAS of the miR-15 and miR-106b families located in the miR-17–92, miR-106a-363, and miR-106b-25 clusters. Transfection experiments demonstrated that the observed downregulation of miR-106a in senescent cells contributed to the upregulation of the regulator of cellular senescence p21CDKN1A.⁶⁰

MiR-204 was also consistently downregulated in both stress induced and replicative senescence in HTM cells. Transfection experiments with miR-204 showed the regulation of multiple genes in HTM cells by this miRNA. Twelve of these genes (AP1S2, BCL2L2, BIRC2, EDEM1, EZR, FZD1, M6PR, RAB22A, RAB40B, SERP1, TCF12, and TCF4) were experimentally validated as direct targets of miR-204. Downregulation of expressions at protein levels of BCL2L2, BIRC2, EZR, M6PR, and SERP1 were confirmed by western blot analysis. Forced expression of miR-204 in HTM cells resulted in increased levels of apoptosis, decreased viability, increased accumulation of oxidized proteins after H₂O₂ treatment, decreased induction of ER stress response markers, and reduced expression of inflammatory mediators IL8 and IL11.⁶² These results suggested that the downregulation of miR-204 observed in senescent cells could potentially contribute to the increased resistance to apoptosis and help to prevent the accumulation of damaged proteins and excessive production of inflammatory mediators characteristic of senescent cells. Recently, it has also been reported that miR-204 caused decreased expression of FOXC1 and the FOXC1 target genes CLOCK, PLEKHG5, ITGB1, and MEIS2 in TM cell cultures.⁶³ The inhibition of FOXC1 by miR-204 is potentially relevant in the TM since mutations in this gene are among the causes of Axenfeld-Rieger syndrome, which is often accompanied by elevated intraocular pressure (IOP).^64,65 These results support the concept that miR-204 might play a relevant role in the TM and that alterations in expression of this miRNA could contribute to pathogenic changes.

Some miRNAs, such as miR-183, were upregulated in SIPS and downregulated in replicative senescence. Analysis of the effects mediated by miR-183 in HTM senescent cells lead to the identification and experimental validation of 2 novel targets: integrin beta1 (ITGB1) and kinesin 2 alpha (KIF2A). MiR-183 significantly decreased the expression of ITGB1 and KIF2A and significantly decreased adhesion to laminin, gelatin, and collagen type I in HTM cells.⁶⁶ These effects were rescued by expression of ITGB1 lacking the 3′-UTR, suggesting that changes in expression of miR-183 could contribute to alterations in the behavior of HTM cells through the inhibition of ITGB1.

Two miRNAs consistently upregulated in replicative senescent HTM cells were miR-146a and miR-146b. These miRNAs are also upregulated during replicative senescence and aging in other cells types including fibroblasts and macrophages.^67–69 Upregulation of miR-146a/b is also associated with several pathological alterations.^70,71 Consistent with the anti-inflammatory effect proposed for miR-146a/b, transfection of HTM cells with miR-146a agomirs resulted in downregulation of multiple genes associated with inflammation, including IRAK1, IL6, IL8, and SERP1. Further, miR-146a inhibited senescence-associated β-galactosidase activity and production of intracellular reactive oxygen species (iROS), and increased cell proliferation. Overexpression of either IRAK1 or SERP1 inhibited the effects of miR-146a on cell proliferation and iROS production in HTM senescent cells.⁶¹ Upregulation of the anti-inflammatory miR-146a in senescent cells has been hypothesized to restrain excessive production of inflammatory mediators and limit their deleterious effects on the surrounding tissue.⁶⁷ Among the different proteins repressed by miR-146a, the inhibition of SERP1 may act to minimize the effects of senescence on the generation of ROS and growth arrest, and prevent alterations of the extracellular proteolytic activity of the TM.

All together, these results suggest that changes in miRNA expression contribute to the phenotypic alterations observed in senescent cells, and in particular those by which the presence of senescent cells in the TM might contribute to alter the physiology of the outflow pathway.

Changes in miRNA Expression Associated with Mechanical Stress

The TM is exposed to constant cycles of stretching and relaxation as a result of blood pressure oscillations associated with systole and diastole, and fluctuations of IOP.^72–75 Mechanical stress has been shown to induce an array of responses in TM cells that could be relevant for the homeostatic regulation of outflow facility and at the same time contribute to pathological alterations of the TM.^76–78 Cyclic mechanical stress can also induce the progression of fibrosis in connective tissues.^79,80 Therefore, our laboratory investigated potential changes in miRNA expression induced by mechanical stress in HTM cells.⁸¹ These studies were conducted in 3 lines of primary cultured HTM cells subjected to cyclic mechanical stress for 3 h at 20% stretching, 1 cycle per second. The results showed consistent upregulation of 7 miRNAs in all cell lines (miR-16, miR-27a, miR-27b, miR-7, let-7f, miR-26a, and miR-24) and 9 miRNAs significantly upregulated in 2 cell lines (miR-28-3p, miR-302b, miR-100, miR-106b, miR-146a, miR-22, miR-126, and let-7a). Interestingly, several of these miRNAs have been implicated in the regulation of fibrosis. Transgenic mice overexpressing miR-27b in the heart have been shown to develop cardiac fibrosis that may result from inhibition of MMP-16.⁸² On the other hand, miR-16 is known to inhibit TGFβ1 release in human alveolar epithelial cells⁸³ and is believed to have anti-fibrotic activity.^84,85 MiR-26 appears to be also anti-fibrotic in the heart by inhibiting collagen I and connective tissue growth factor.⁸⁶ Inhibition of the let-7 family leads to changes consistent with epithelial mesenchymal transition in lung epithelial cells both in vitro and in vivo.⁸⁷ The let-7 family is believed to inhibit fibrosis by repressing expression of collagen genes.⁸⁸ Finally, experiments conducted in our laboratory identified the subtilisin-like proprotein convertase FURIN, which is known to play a major role in the processing of TGFβ1, as a direct target of miR-24 in HTM cells.⁸¹ Consistent with the targeting of FURIN by miR-24, overexpression of miR-24 resulted in a significant decrease in activated TGFβ1, and, conversely, inhibition of miR-24 expression with a specific antagomir led to a small but significant increase in TGFβ1. The effects of miR-24 on the expression of activated TGFβ1 were mimicked by siRNA mediated downregulation of FURIN in HTM cells. MiR-24 is downregulated in the heart after myocardial infarction, and this change in miR-24 expression is inversely correlated with cardiac fibrosis. Administration of miR-24 to the heart with lentiviral vectors has been demonstrated to improve heart function and attenuate fibrosis in the infarct border zone of the heart 2 weeks after induction of myocardial infarction potentially through inhibition of the furin-TGFβ1 pathway.⁸⁹

The observed upregulation of multiple miRNAs involved in the regulation of fibrotic responses suggests that the balance between induction of pro-fibrotic (miR-27) and anti-fibrotic miRNAs (let-7, miR-24, miR-16, and miR-26) could play an important role in preventing the development of fibrosis in the TM as a result of the constant cycles of stretching and relaxation that affect this tissue. Therefore, an imbalance in the expression of these pro- and anti-fibrotic miRNAs could potentially contribute to pathological alterations of the TM leading to increased outflow resistance in glaucoma. Restoring this balance could potentially constitute a new therapeutic approach to delay the functional alteration of the TM in glaucoma.

Regulation of Contractility by miRNAs in TM Cells

Abundant evidence demonstrate that inhibition of the actomyosin system of the outflow pathway cells effectively increases aqueous humor drainage and lower IOP.^90–92 To identify miRNAs involved in the regulation of the actomyosin system in the TM, we conducted a preliminary screening measuring the effects of several miRNAs on HTM cells using a collagen contractility assay. The results showed that at least some miRNAs have the ability to significantly inhibit HTM cells contraction induced by serum (Fig. 1). These results also pointed at miR-200c as an important modulator of the contractile responses in HTM cells.⁹³ MiR-200c is known to regulate epithelial to mesenchymal transitions through repression of the transcription factors ZEB1 and ZEB2 in several cell types.⁹⁴ miR-200c also targets the regulator of stress fibers formation FHOD1.⁹⁵ We found that in HTM cells, in addition to regulating the expression of these 3 genes, miR-200c directly inhibits the expression of RhoA kinase and a number of effectors of the Rho-ROCK cascade including endothelin receptor type A (EDNRA) and lysophosphatidic acid (LPA) receptor 1 (LPAR1).⁹³ Both ET-1 and LPA are present in the aqueous humor and decrease aqueous humor outflow facility by mechanisms dependent on cell contraction mediated by Rho kinase signaling.

FIG. 1.

Effects of selected microRNAs (miRNAs) on the contraction of human trabecular meshwork (HTM) cells in collagen gels. HTM cells were transfected with miRNA mimics or scrambled controls embedded in collagen gels and exposed to culture media with serum. Inhibition of cell contraction mediated by each miRNA was calculated based on the difference between the gel surface compared to controls. miR-204, miR-155, and miR-200c significantly inhibited HTM cell contraction (n=3, t-test, *P<0.01).

Consistently, miR-200c potently inhibited the contraction of TM cells induced by serum, thrombin, endothelin, LPA, and the glaucoma pathogenic factor TGFβ2. MiR-200c also demonstrated a significant effect on IOP in vivo. Hence, administration of 2 consecutive intracameral injections of liposomes carrying miR-200c agomirs to living rats resulted in a significant decrease in IOP despite using a relatively low efficient method for in vivo delivery. In addition, inhibition of miR-200c by intracameral infusion of an adenoviral vector expressing a miR-200c molecular sponge (a transcript with repeated miRNA antisense sequences that sequesters specific miRNAs from their endogenous targets)⁹⁶ lead to a robust IOP increase in living rats.

All together, these results demonstrate the ability of miRNAs to regulate contractility in TM cells and modulate IOP in vivo. More comprehensive studies should help to identify additional miRNAs involved in the regulation of the actomyosin system in the TM and their potential as therapeutic targets in glaucoma.

Regulation of ECM by miRNAs in TM Cells

Regulation of ECM synthesis and degradation is believed to play particularly important roles in outflow physiology and in the pathogenesis of the outflow pathway in glaucoma.^97–99 Changes in the quality and amount of the ECM in the juxtacanalicular region of the TM and the basement membrane of SC appear to be a causative factor in the abnormal increase in aqueous humor outflow resistance in glaucoma.^100,101 Given the importance of ECM dynamics on the normal physiology of the outflow pathway, miRNAs that regulate ECM metabolism appear as likely candidates to influence aqueous humor resistance.

Probably the best characterized family of miRNAs that regulates ECM metabolism is miR-29. The 3 main mature miRNAs processed from these precursors are known as hsa-miR-29a, hsa-miR-29b, and hsa-miR-29c. In humans, miR-29a and miR-29b-1 are processed from an intron of a long non-coding transcript (LOC646329) from chromosome 7. MiR-29b-2 (identical in sequence to miR-29b-1) and miR-29c are co-transcribed from chromosome 1. The miR-29 family members share a common seed region sequence and are predicted to target largely overlapping sets of genes. Each miR-29 directly target multiple genes involved in ECM composition and regulation, providing one of the best examples of targeting a large group of functionally related genes by a single miRNA. Consistent with their effects on ECM metabolism, miR-29s have strong antifibrotic effects in heart, kidney, and other organs.¹⁰² Interestingly, genes with predicted target sites for the miR-29 family are among the more abundant non-housekeeping genes expressed in TM cells.¹⁰³

The effects of miR-29s on HMT cells were first investigated by gene array analysis.¹⁰⁴ Transfection of HTM cells with miR-29b mimic resulted in downregulation of multiple ECM components, including collagens (COL1A1, COL1A2, COL4A1, COL5A1, COL5A2, and COL3A1) LAMC1, and FBN1 and several genes involved in ECM deposition and remodeling, such as SPARC. In these studies, 3 additional genes, BMP1, ADAM12, and NKIRAS2, were identified and experimentally validated as direct targets of miR-29b. Interestingly, chronic oxidative stress induced by incubation at 40% oxygen lead to a significant downregulation of miR-29b in 2 HTM cell lines that was associated with increased expression of several ECM genes known to be regulated by miR-29b. The increase in expression of these genes was inhibited by transfection with miR-29b mimic. In addition, miR-29b increased cell viability under both chronic oxidative stress and physiologic oxygen concentrations.

Because of the well-known role of TGFβ signaling on ECM regulation and in particular the potential pathogenic role of TGFβ2 elevation in POAG,^105,106,107 the relationship between TGFβs and miR-29s in HTM has been investigated by us and Villarreal et al. (Fig. 2).^108,109

FIG. 2.

Working models for the interaction of miR-29s and TGFβs based on the studies conducted by Villareal et al.¹⁰⁹ (A) and Luna et al.¹⁰⁸ (B). In both models miR-29s inhibit extracellular matrix (ECM) synthesis and can antagonize the effects of TGFβ2.

In our laboratory, we found that TGFβ2 downregulated the 3 members of the miR-29 family and that TGFβ1 had no effects on the expression of these miRNAS.¹⁰⁸ In contrast, Villarreal et al. found that, although all 3 members of the miR-29 family were expressed in cultured TM cells, incubation with TGFβ2 induced miR-29a and suppressed miR-29b levels.¹⁰⁹ Transfection of miR-29b mimics antagonized the effects of TGFβ2 on the expression of several ECM components, suggesting that the downregulation of miR-29 by TGFβ2 contributed to the induction of several ECM components by this cytokine in HTM cells. In addition, miR-29b decreased the expression of TGFβ1 at the promoter, transcript, and protein levels but had only a minor effect on the expression of active TGFβ2. The inhibition of TGFβ1 by miR-29b was partially recovered after co-transfection with a plasmid-expressing BMP1.¹⁰⁸ Villareal et al. showed that SMAD3 modulates miR-29b expression under basal and TGFβ2 conditions. Subsequent gain- and loss-of-function experiments conducted by the same authors confirmed the inhibition of various ECM proteins by miR-29s family under basal and TGFβ2 stimulatory conditions.¹⁰⁹

All together these studies demonstrated that the miR-29 family is a critical regulator of ECM expression in the TM, and suggested that downregulation of miR-29b might contribute to increased expression of ECM genes induced by chronic oxidative stress and TGFβ2.

Future Developments and Concluding Remarks

The limited information generated so far about the role of miRNAs in the TM suggests that these regulatory elements can modulate an array of cellular functions relevant to the physiology and pathophysiology of the outflow pathway (Table 1). However, to realize the full potential of miRNAs as diagnostic and therapeutic targets in glaucoma it will be necessary to fill up major gaps in our current knowledge. Some of the important needs to better understand the biology of miRNAs in the TM include the following.

Table 1.

Summary of the miRNAs Investigated in Trabecular Meshwork Cells

miRNA ID (accession)	Expression changes in HTM cells	Genes downregulated in HTM cells	Cellular functions	References
hsa-miR-24-3p (MIMAT0000080)	Induced by mechanical stress	FURIN	TGFβ1 processing	43
hsa-miR-29b-3p (MIMAT0000100)	Downregulated by TGFβ2 and chronic oxidative stress	COL1A1, COL1A2, COL4A1, COL5A1, COL5A2, COL3A1, LAMC1, FBN1, SPARC, BMP1, ADAM12, NKIRAS2, SP1	Extracellular matrix metabolism and TGFβ signaling	58,60,61
hsa-miR-106a-5p (MIMAT0000103)	Downregulated in senescence	p21CDKN1A	Cell proliferation, apoptosis, and senescence	33
hsa-miR-146a-5p (MIMAT0000449)	Upregulated in replicative senescence and mechanical stress	IRAK1, IL6, IL8, SERP1, CCL2 CXCL3 IL11, SLC10A3, PTGS1, GLANT10, CCL20, CXCL6, PPP2R1B, HAS1	Retinoic acid receptor and DNA damage response	38
hsa-miR-182-5p (MIMAT0000259)	Upregulated in stress induced senescence	RARG	Cell proliferation, DNA damage response, and ITGB1 signaling	33
hsa-miR-183-5p (MIMAT0000261)	Upregulated in stress induced senescence	ITGB1, KIF2A	Production of inflammatory mediators	37
hsa-miR-200c-3p (MIMAT0000617)	Upregulated in stress induced senescence	ZEB1, ZEB2, FHOD1, EDNRA, LPAR1, RHOA	Cell contraction	53
hsa-miR-204-5p (MIMAT0000265)	Downregulated in stress induced and replicative senescence	AP1S2, BCL2L2, BIRC2, EDEM1, EZR, FZD1, M6PR, RAB22A, RAB40B, SERP1, TCF12, TCF, FOXC1, CLOCK, PLEKHG5, ITGB1 MEIS2	Regulation of apoptosis, endoplasmic reticulum stress response, and inflammation	35,36

miRNA, microRNA; HTM, human trabecular meshwork.

Comprehensive identification miRNAs expressed in the TM at the tissue level

So far, only miRNA expression analysis has been conducted in primary cultured cells that do not reflect the actual expression profile of the tissue. Based on the findings in other age-related diseases it is likely that the miRNA expression profile may be altered in glaucoma, aging, and other stress conditions such as elevated IOP. Information about these potential changes would be valuable to identify specific miRNAs relevant to both the normal physiology and the pathology of the TM.

Characterization of miRNA specific functions at the cellular level

Although valuable information regarding target genes and functional effects can be extrapolated from other tissues, miRNAs do not target the same sets of genes in each cell type or exert the same functional effects since such effects are dependent on the specific target genes that are expressed in each particular cell type. In addition, perhaps as many as 16% of pre-miRNAs may be altered through nuclear RNA editing. Tissue-specific editing of a specific miRNA can alter its target specificity by changing the seed region. Finally, tissue-specific alternative polyadenylation sites of a gene can alter the presence of miRNA target sites present in the transcript, thus changing the miRNAs that can regulate its expression in different tissues. Therefore, conducting experiments in TM cells is a necessary step to identify the specific gene targets and biological effects of a given miRNA in the TM.

Profiling of extracellular miRNAs

The study of miRNAs present in serum and other biological fluids has already provided valuable information for diagnosis and prognosis of several diseases. The interest in miRNAs as biomarkers is growing as more and more evidence supports that miRNAs are actively secreted from diseased tissues, possibly before the onset of overt disease.³⁹ However, there are no reports about alterations in miRNAs present in either aqueous humor or plasma associated with glaucoma. Such studies have the potential to identify novel biomarkers for early diagnosis of glaucoma. Similarly, the export of miRNAs to the extracellular space has not been investigated in TM cells. Intercellular communication is likely to play an important role in the normal and pathologic physiology of the outflow pathway. Although the investigation of extracellular miRNAs in cell–cell signaling is in its early stages, there is increasing evidence that many cells actively secrete miRNAs in a selective manner¹¹⁰ and that intercellular transport of miRNAs can exert physiological effects in adjacent cells.^111,112 The study of extracellular miRNAs produced by TM cells under different conditions could provide insights about how TM cells can influence the behavior of adjacent cells including those of the SC.

Development of methods for in vivo delivery of miRNAs to the cells of the TM

Current methods for in vivo delivery of miRNAs include the direct administration of chemically modified agomirs with 2-O-methyl-group or 2-O-methyoxyethyl or using LNAs to increase drug stability and affinity. A variety of carriers have been developed to increase delivery efficiency including lyposomes, nanoparticles, and microvesicles. However, these methods provide only a short-term increase in miRNA expression. To achieve longer-term expression it may be necessary to use viral-based vectors. Although vectors derived from DNA viruses can be used, they tend to compete with the cellular pathways of miRNA biogenesis creating unwanted alterations in the expression of endogenous miRNAs and some toxicity. Novel vectors using non-conventional pathways to express exogenous miRNAs are being developed to circumvent this problem. Because of the small size of miRNAs, some of these vectors can express multiple miRNAs (at least up to 10) in a single carrier (TenOevier, pers. comm.). This characteristic provides a unique opportunity to design gene therapy agents combining the biological effects of different miRNAs to achieve synergistic and/or additive therapeutic effects. Some promising miRNAs include the miR-29s, miR-200c, miR-204, and miR-24 that regulate functions implicated in the pathogenesis of POAG such as ECM deposition, cell contraction, ER stress, apoptosis, inflammation, fibrosis, and TGFβ1 processing.

In conclusion, miRNAs offer an attractive opportunity to identify novel biomarkers for diagnosis and prognosis of glaucoma and to develop therapeutic agents capable of simultaneously modifying different cellular functions affected in POAG for personalized treatment of this multifactorial disease. Future studies aimed at filling up the current gaps of knowledge about miRNAs in the TM should help to understand their roles in the outflow pathway and exploit their full potential as diagnostic and therapeutic targets for glaucoma. Specifically, the ability of miRNAs to simultaneously regulate multiple aspects of the cellular physiology might offer a unique opportunity to modify the behavior of TM cells in glaucoma patients to restore functionality of the outflow pathway.

Acknowledgments

Supported by National Eye Institute Grants EY01894, EY016228, EY023287, and EY05722 and by Research to Prevent Blindness.

Author Disclosure Statement

No competing financial interests exist.