Extracellular matrix in the trabecular meshwork

The extracellular matrix (ECM) of the trabecular meshwork (TM) is thought to be important in regulating intraocular pressure (IOP) in both normal and glaucomatous eyes. IOP is regulated primarily by a fluid resistance to aqueous humor outflow. However, neither the exact site nor the identity of the normal resistance to aqueous humor outflow has been established. Whether the site and nature of the increased outflow resistance, which is associated with open-angle glaucoma, is the same or different from the normal resistance is also unclear. The ECMs of the TM beams, juxtacanalicular region (JCT) and Schlemm's canal (SC) inner wall are comprised of fibrillar and non-fibrillar collagens, elastin-containing microfibrils, matricellular and structural organizing proteins, glycosaminoglycans (GAGs) and proteoglycans. Both basement membranes and stromal ECM are present in the TM beams and JCT region. Cell adhesion proteins, cell surface ECM receptors and associated binding proteins are also present in the beams, JCT and SC inner wall region. The outflow pathway ECM is relatively dynamic, undergoing constant turnover and remodeling. Regulated changes in enzymes responsible for ECM degradation and biosynthetic replacement are observed. IOP homeostasis, triggered by pressure changes or mechanical stretching of the TM, appears to involve ECM turnover. Several cytokines, growth factors and drugs, which affect the outflow resistance, change ECM component expression, mRNA alternative splicing, cellular cytoskeletal organization or all of these. Changes in ECM associated with open-angle glaucoma have been identified.     

小梁网的干细胞移植治疗原发性开角型青光眼的研究进展

原发性开角型青光眼(primary open angle glaucoma,POAG)是以持续性眼压增高导致视神经损伤为主要临床表现的一种疾病,其发病机制复杂,尚未明确,现阶段临床治疗相对困难。影响眼内压(intraocular pressure,IOP)高低的重要因素是房水引流是否通畅,而房水引流途径中小梁网(trabecular meshwork,TM)起重要调控作用。TM细胞的形态、数量、结构和功能改变均可使房水外流阻力增大,从而导致IOP升高。研究证实诱导多功能干细胞(induced pluripotent stem cells,iPSCs)、骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)和脂肪干细胞(adipose-derived stem cells,ADSCs)已被用于TM细胞的分化和再生,为POAG小梁网的干细胞替代治疗提供可靠的细胞来源。近年研究发现,小梁网干细胞(trabecular meshwork stem cells,TMSCs)在分化为TM细胞方面具有绝对优势,为细胞移植治疗青光眼提供新的靶向,这标志着干细胞治疗POAG进入一个新纪元,为青光眼治疗带来新的曙光。本文将对不同种类干细胞的小梁网移植进行综述,为细胞移植治疗POAG提供新思路。     

The matricellular protein SPARC is expressed in human trabecular meshwork

PURPOSE: This investigation was undertaken to determine whether the matricellular protein SPARC is expressed in the human trabecular meshwork (TM) and cultured human trabecular meshwork cells. METHODS: Human donor trabecular meshwork and cultured cells obtained from trabecular meshwork were used in this study. Total RNA was obtained from TM and cultured TM endothelial cells, and RT-PCR was done with primers specific for SPARC. Western blotting was performed on donor TMs using an anti-SPARC monoclonal antibody prepared against rHuSPARC. Confocal microscopy was used to determine the distribution of SPARC in human anterior segments, and immunofluorescence on cultured TM cells was performed with the anti-SPARC antibody. RESULTS: SPARC mRNA was expressed both in TM and in cultured TM cells. Immunoblotting for SPARC showed a doublet with a molecular mass approximately 43 kDa. The ratio of the doublet bands varied with each of the samples; some of the cultured cells and the tissue samples exhibited more of the upper band, and other cultured cells contained almost equal amounts of the two bands. The upper band was shown to be a glycosylated form of SPARC. Immunofluorescence showed that SPARC was expressed in the cultured TM, and confocal microscopy with the anti-SPARC antibody demonstrated the presence of this protein in the TM and in other tissues in the anterior segment. CONCLUSIONS: Our data conclusively show that SPARC mRNA and protein are present in non-glaucomatous TM tissue and in cultured TM cells. Because of its effect on matrix metalloproteinases, SPARC may play a role in the regulation of intraocular pressure.     

A review of genetic and structural understanding of the role of myocilin in primary open angle glaucoma

Primary open angle glaucoma (POAG) is the most common form of glaucoma and the second leading cause of blindness in the world. Discovery of the candidate gene MYOC (TIGR/MYOC) encoding the protein myocilin, believed to have a role in cytoskeletal function, might play a key role in understanding the pathogenesis of POAG. MYOC is expressed in many ocular tissues, including trabecular meshwork (TM), a specialised eye tissue essential in regulating intraocular pressure (IOP). Later it was shown to be the trabecular meshwork inducible-glucocorticoid response protein (TIGR). Mutations in MYOC have been identified as the cause of hereditary juvenile-onset open-angle glaucoma (JOAG). The unprocessed myocilin with signal peptide is a 55-kDa protein with 504 amino acids. Mature myocilin is known to form multimers. Wild type myocilin protein is normally secreted into the trabecular extracellular matrix (ECM) and there appears to interact with various ECM materials. It is believed that the deposition of high amounts of myocilin in trabecular ECM could affect aqueous outflow either by physical barrier and/or through cell-mediated process leading to elevation of IOP. The N-terminal region of the myocilin has sequence similarity to myosin (muscle protein) and the C-terminal of the protein has an olfactomedin-like domain. Structural and genetic studies of the MYOC gene and its protein product along with molecular modeling could lead to better understanding of the pathogenesis of POAG. This review highlights the current understanding of myocilin and the relevance of genetic and structural work.     

Recent developments in understanding the pathophysiology of elevated intraocular pressure

PURPOSE OF REVIEW: The state of the actin cytoskeleton and adhesions of trabecular meshwork cells are important determinants of fluid outflow through the trabecular meshwork. Dysregulation of these subcellular structures or cell loss itself, is expected to adversely affect aqueous humour dynamics and intraocular pressure. This article reviews recent research into the regulation of the cytoskeleton and cell adhesions within the trabecular meshwork. RECENT FINDINGS: Key cytoskeleton regulatory pathways in trabecular meshwork cells and their extracellular matrix significantly influence outflow facility. Integrins and matrix proteins play an important part in cell-matrix communication and mediate trabecular meshwork cytoskeletal changes. Increased cross-linking of the actin cytoskeleton may render the trabecular meshwork stiffer and more resistant to aqueous outflow. In-vitro studies show that transforming growth factor-beta induces actin stress fibres in trabecular meshwork cells, indicating that the cells become more contractile. Myocilin and the heparin II domain of fibronectin also influence the actin cytoskeleton. Mutated myocilin appears to affect trabecular meshwork cells differently from wild-type myocilin and can reduce cell survival. Reduced cell survival is also associated with primary open angle glaucoma, ageing, cellular senescence and oxidative insults. SUMMARY: These findings represent advances in understanding physiological and pathogenic mechanisms within the trabecular meshwork that are relevant to intraocular pressure regulation in health and glaucoma. They pave the way for future research on the pathogenesis of glaucoma and new targets for glaucoma therapy.     

小梁网泵调控理论及其对青光眼诊疗的启示

小梁网房水引流功能异常导致眼压升高,是原发性开角型青光眼发生发展的最主要危险因素。房水具有搏动外流的特征,说明小梁网是弹性组织,其弹性状态与Schlemm管腔开放程度、管类瓣膜结构联动和深层巩膜静脉丛的开放与闭合密切相关,表明小梁网对房水的引流具有泵调控机制。本文对小梁网泵调控机制的理论基础、相关检查的临床应用及对青光眼微创手术的启示进行了系统介绍。希望小梁网泵调控理论为了解原发性开角型青光眼发病机制、选择治疗策略、优化和创新手术设计提供新的视角。(眼科,2022,31:405-412)     

Impact of the clinical use of ROCK inhibitor on the pathogenesis and treatment of glaucoma

Rho-associated protein kinase (ROCK), a ubiquitously expressed signaling messenger and downstream effector of Rho, is activated by several bioactive factors in the aqueous humor (AH). Rho-ROCK signaling regulates a wide spectrum of fundamental cellular events, including cell adhesion, motility, proliferation, differentiation, and apoptosis. Previous studies, including our own, found that ROCK inhibitor lowers intraocular pressure (IOP) via a direct effect on the conventional AH outflow pathway, by regulation of contractile properties, fibrotic activity, and permeability of the trabecular meshwork (TM) and Schlemm’s canal (SC) tissues, influencing extracellular matrix (ECM) production. Recently, a novel ROCK inhibitor, ripasudil, has been introduced in Japan. Other ROCK inhibitors are now in clinical trials as new IOP-lowering drugs for glaucoma patients. To date, ripasudil, administered together with other glaucoma medications, has proved safe and efficient in lowering IOP as well as additional effects such as prostaglandin analogs, beta-blockers, and carbonic anhydrase inhibitors, all of which help lower IOP by different mechanisms. In addition, we found that long-term treatment with ripasudil exerted an additional IOP-lowering effect, especially in eyes with high IOP, suggesting that late-onset remodeling of the ECM in glaucomatous eyes may elicit mild and delayed changes in IOP levels. ROCK inhibitors have also shown several additional effects, including increased retinal blood flow, direct protection of neurons against various types of stress, and regulation of wound healing; these benefits may potentially be useful in glaucoma treatment.     

原发性闭角型青光眼小梁细胞相关变化的研究进展

原发性闭角型青光眼房角关闭致房水流出受阻,眼压升高。随着房角的关闭,小梁细胞在形态及生理功能上都会发生变化。有报道原发性闭角型青光眼经超声乳化联合房角分离术后,在一定时间内眼压会有不同程度的下降,说明房角重新开放后,小梁细胞也发生了相关变化,功能得到了不同程度的恢复。但其具体机制以及与房角关闭持续时间的关系等还需要进一步研究和探讨。     

Modulation of extracellular matrix turnover in the trabecular meshwork

Intraocular pressure (IOP) is the most critical risk factor for primary open angle glaucoma (POAG). In most cases of POAG, IOP is increased because of an abnormally high aqueous humor outflow resistance in the juxtacanalicular region of the trabecular meshwork. A distinct structural change in the trabecular meshwork of patients with POAG is the increase in fibrillar extracellular matrix in the juxtacanalicular region of the trabecular meshwork. Our knowledge on the molecular factors that govern turnover of the extracellular matrix in the trabecular meshwork has increased considerably in recent years. It has become clear that quality and quantity of the extracellular matrix in the trabecular meshwork are regulated by several signaling molecules that interact with each other to promote its synthesis, degradation, or extracellular modification. Transforming growth factor-β1 and β2 (TGF-β1 and TGF-β2) which derive from the aqueous humor or may be locally expressed induce in cultured trabecular meshwork cells the expression of a variety of extracellular matrix molecules. The action of TGF-βs very likely requires local activation by thrombospondin-1 and is partly mediated by its downstream mediator connective tissue growth factor, both of which are constitutively expressed in the trabecular meshwork. Bone morphogenetic proteins (BMP)-7 and -4 effectively antagonize the effects of TGF-β2 on matrix deposition. The antagonizing effects of BMP-7 are mediated in trabecular meshwork cells through Smad7. Smad7 is a key molecular switch to inhibit TGF-β2 signaling in the trabecular meshwork.     

The vital role for nitric oxide in intraocular pressure homeostasis

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

The trabecular meshwork in acute and chronic angle closure glaucoma

PURPOSE: To determine the effect of acute and chronic primary angle closure glaucoma (PACG) on the trabecular meshwork. METHODS: Trabecular specimens of 16 consecutive patients with primary angle closure glaucoma (PACG)--6 acute PACG eyes, and 10 chronic PACG eyes without an acute attack--were studied by light and electron microscopy. RESULTS: Acute PACG: The trabecular meshwork revealed a generalised oedema and an accumulation of pigment in the widened trabecular spaces and Schlemm's canal. Attenuated trabecular endothelial cells appeared to be devoid of subcellular components. Chronic PACG: In chronic PACG eyes the trabecular architecture had lost its regular arrangement, with fewer and narrower trabecular spaces and fusion of the trabecular beams in areas. There were numerous electron-dense bodies in the trabecular tissues, both within the trabecular beams and in the extracellular spaces, which had a banded fibrillar structure. An overall loss of endothelial cells was noted; the remaining cells were crowded together and were polymorphic. Melanin pigment was present both within the stroma and in the endothelial cells. CONCLUSIONS: Pigment accumulation in the trabecular spaces and within the cells and a noninflammatory degeneration appeared to be the primary changes in the trabecular meshwork after acute angle closure glaucoma. In chronic PACG eyes, there was evidence of loss of endothelial cells and reactive repair processes. These changes were present in areas away from visible peripheral anterior synechiae. A gonioscopic evaluation of the extent of peripheral anterior synechiae alone may not reflect the extent of trabecular meshwork damage in acute and chronic PACG. Patients experiencing an acute attack of PACG require a long-term follow up, because the intraocular pressure (IOP) may rise later, due to ongoing changes compromising the outflow facility, or due to the effects of aging in the trabecular meshwork.     

水通道蛋白-1在小梁切除术之切除组织中的表达

目的观察青光跟患者小梁和虹膜组织与正常跟组织水通道蛋白-1（AQP-1）的表达差异。方法收集开角型和闭角型青光跟小梁切除术时切除的小梁和虹膜组织，免疫组织化学法检测AQP-1的表达，并与正常跟相应组织对照。结果正常眼小梁网组织、Schlemm’s管内皮细胞、周边虹膜组织中上皮和基质组织可见AQP-1呈强阳性着色，开角型青光眼和闭角型青光眼组织标本小梁网AQP-1阳性染色较正常弱；部分急性闭角型青光眼患者周边虹膜组织标本上皮层较基质组织染色明显弱。结论开角型青光眼小梁网AQP-1的表达减少可能与小梁网的发育有关，闭角型青光眼虹膜上皮和小梁网AQP-1的表达减少可能与虹膜萎缩或高眼压有关。     

细胞外基质重塑在原发性开角型青光眼的作用及研究进展

青光眼是一组以特征性视神经萎缩和视野缺损为共同特征的疾病,病理性眼压增高是其主要危险因素。视网膜神经节细胞( retinal ganglion cells, RGCs)凋亡及其轴突丢失是青光眼的主要病理特征。细胞外基质( extracellular matrix, ECM)含量和成分的变化对小梁网构型、视乳头筛板结构、RGCs凋亡起着决定性作用。青光眼患者小梁网及房水中转化生长因子-β2( transforming growth factor-β2,TGF-β2)增加,引起ECM分泌增加和堆积导致眼压升高;高眼压引起视神经乳头ECM成分的改变,引起神经营养因子剥夺,导致RGCs凋亡;同时,高眼压引起视网膜基质金属蛋白酶类-9(matrix metalloproteinase-9,MMPs-9)活性增加,层连黏蛋白的减少又将导致 RGCs 凋亡的增加。因此,研究ECM和青光眼的关系至关重要,可能为原发性开角型青光眼发病机制及治疗提供新的方向。     

Baseline IOP predicts selective laser trabeculoplasty success at 1 year post-treatment: results from a randomised clinical trial

BACKGROUND/AIMS: The efficacy and safety of selective laser trabeculoplasty (SLT) has been found to be equivalent to argon laser trabeculoplasty (ALT). Since SLT produces significantly less disturbance to the trabecular meshwork and is theoretically more repeatable than ALT, it has potential to replace ALT as the standard procedure to treat medically uncontrolled open angle glaucoma. This study's objective is to determine factors that predict successful SLT at 1 year post-treatment. METHODS: As part of a randomised clinical trial comparing the efficacy and safety of SLT to ALT, data on 72 SLT patients were collected, and successful SLT defined as having an SLT induced intraocular pressure (IOP) reduction of >or=20% at 1 year post-treatment follow up. RESULTS: 43 out of the 72 patients who had completed their 1 year follow up visit had an IOP reduction of >or=20% from baseline. No glaucoma risk factors studied predicted successful SLT. The amount of trabecular meshwork pigmentation was not a significant predictor. However, it was discovered that baseline IOP strongly predicted SLT success (odds ratio=1.16; p=0.0001). CONCLUSION: SLT success was significantly predicted by baseline IOP but not by age, sex, other glaucoma risk factors, type of open angle glaucoma, or by degree of trabecular meshwork pigmentation.     

Proteins secreted by human trabecular cells. Glucocorticoid and other effects

The capacity of cultured human trabecular meshwork (HTM) cells to secrete an extracellular matrix was studied by indirect immunofluorescence. Synthesis of nine extracellular matrix (ECM) proteins known to be present in the normal trabecular meshwork was assessed in three HTM cell lines. Fourteen primary antibodies were used and cultures were labeled two and four weeks after confluence. The HTM cell lines showed consistent labelling patterns for the normal extracellular connective tissue constituents including collagens (types I, III, IV, V and VI), glycoproteins (laminin and fibronectin) and a basement membrane-associated proteoglycan. These antigens were localized to the basal cell surface in an extracellular reticular pattern corresponding to cell margins. Dextran addition at confluence helped to intensify the staining of these components, but ascorbate had no apparent effect. Interestingly, elastin, another normal component of the trabecular meshwork, was not identified under standard conditions, or after addition of ascorbate or dextran. However, elastin could be detected intracellularly following dexamethasone treatment for three days, and extracellularly in punctate deposits when this treatment was used for 1 or 2 weeks. Our findings indicate that HTM cells may be responsible for the secretion and maintenance of all the major ECM constituents of the trabecular meshwork. The elastin results suggest a possible mechanism contributing to obstruction of outflow in steroid glaucoma if increased amounts of elastin are also produced in vivo. This approach can also serve as a useful baseline for comparison with HTM cell lines treated with glaucoma medications or obtained from patients with glaucoma.     

Transforming growth factor-beta2 utilizes the canonical Smad-signaling pathway to regulate tissue transglutaminase expression in human trabecular meshwork cells

Transforming growth factor-beta2 (TGF-β2) is elevated in the aqueous humor of patients with glaucoma. This growth factor is known to increase extracellular matrix (ECM) deposition in the trabecular meshwork (TM) as well as increase intraocular pressure (IOP) in perfused human cultured anterior eye segments. In addition overexpression of TGF-β2 in the mouse TM leads to elevated IOP. Exogenous TGF-β2 also increases tissue transglutaminase (TGM2) protein levels and enzyme activity in TM cells. TGM2 is a calcium-dependent enzyme that mediates cross-linking of ECM proteins, thus making ECM proteins resistant to enzymatic degradation and physical breakdown. We have investigated the signaling pathway by which TGF-β2 induces TGM2 in human TM cells. Primary cultures of human TM cells (N = 6) were treated for 48 h with TGF-β2 (0–10 ng/ml) in serum-free medium. TGM2 enzyme activity differences between non-treated and TGF-β2 treated TM cells were studied using a biotin cadaverine assay. Endogenous TGF-β2 protein levels were examined in normal trabecular meshwork (NTM) and glaucomatous trabecular meshwork (GTM) cell strains. Immunohistochemistry was used to evaluate the expression and co-localization of TGF-β2 and TGM2 in NTM and GTM tissues. Activation of Smad3 signaling pathway was evaluated by western immunoblot analysis using phospho-specific antibodies following exogenous TGF-β2 treatment. Pharmacological specific inhibitor of Smad3 (SIS3) and short interfering (si)RNAs were used to suppress Smad3 activity and CTGF gene expression respectively. Endogenous TGF-β2 levels were significantly elevated in cultured GTM cells (p < 0.05) when compared to NTM cells. Immunohistochemistry studies also demonstrated elevated expression and co-localization of both TGF-β2 and TGM2 in glaucoma human TM tissues. Exogenous TGF-β2 increased both TGM2 protein levels and enzyme activity in TM cells. Phosphorylation of Smad3 was stimulated in TM cell strains by exogenous TGF-β2. TGF-β2 induction of TGM2 was not inhibited with selective siRNA knockdown of CTGF. In contrast, a specific inhibitor of Smad3 (SIS3) and siRNA knockdown of Smad3 (p < 0.05) suppressed TGF-β2 induction of TGM2. This study demonstrated that TGF-β2 induction of TGM2 can be mediated via the canonical Smad-signaling pathway but does not appear to involve CTGF as a downstream mediator. Regulation of the Smad-signaling pathway in the TM may be useful in the therapy for glaucoma associated with aberrant TGF-β2 signaling.     

The role of steroids in outflow resistance

Glucocorticoid (GC)-induced ocular hypertension and secondary iatrogenic open-angle glaucoma are serious side effects of GC therapy. Its clinical presentation is similar in many ways to primary open-angle glaucoma, including increased aqueous outflow resistance and morphological and biochemical changes to the trabecular meshwork (TM). Therefore, a large number of studies have examined the effects of GCs on TM cells and tissues. GCs have diverse effects on the TM, altering TM cell functions, gene expression, extracellular matrix metabolism, and cytoskeletal structure. Some or all of these effects may be responsible for the increased outflow resistance associated with GC therapy. In contrast to GCs, several different classes of steroids appear to lower IOP. Additional research will help better define the molecular mechanisms responsible for GC-induced ocular hypertension and steroid-induced IOP lowering activity.     

The psychology of the glaucoma patient

Under physiological conditions, intraocular pressure (IOP) is controlled by the autonomic and central nervous systems. Correspondingly, nerve fibers and neurotransmitters are present in ciliary body and trabecular meshwork. IOP responds to physical as well as psychological stimuli in healthy individuals. In patients with dysregulated IOP, e.g., in those with primary open-angle glaucoma, emotional instability without a specific personality pattern could be found. Whereas the statistical association between emotional changes and glaucoma is obvious, the causal relationship remains to be clarified. It is at least plausible that psychic stress may have an influence on IOP. However, the sequence of the events is unknown (emotional disturbance can be the result of the disease or it can be a primary sign of a nervous dysfunction). Therefore, it seems meaningful--in addition to standard glaucoma therapy--to try to improve the patient's emotional condition, both for treatment of the glaucoma and for the patient's general quality of life.     

非编码RNA调控小梁网组织在青光眼中作用的研究进展

小梁网是前房水引流的重要通道,呈复杂的三维结构,主要的细胞成分是小梁网细胞,细胞之间有交错成多层的细胞外基质。小梁网结构和功能的改变是导致眼压失常甚至视神经损伤的重要原因。有研究发现,非编码RNA(ncRNA)的异常表达可导致小梁网细胞生存率、收缩性能和细胞外基质结构发生改变,房水流出受阻,眼压失控,是青光眼发生的重要机制。目前,与小梁网相关的ncRNA研究涉及多种ncRNA分子和多种类型青光眼,本文将对微小RNA、长链ncRNA和环状RNA等ncRNA在小梁网组织中的研究进展进行综述。