Toxic effects of mitomycin-C on cultured ciliary process cells and trabecular meshwork cells.

Mitomycin-C has recently become an adjunct medication for inhibition of fibroblast proliferation in glaucoma filtering procedures. Prolonged postoperative ocular hypotony has been a frequent complication of trabeculectomy with mitomycin-C. In order to characterize the hypotony mechanism, we compared the toxic effects of mitomycin-C on cultured rabbit ciliary process cells and trabecular meshwork cells. The results indicate that mitomycin-C has a more marked effect on ciliary process cells on 3H-thymidine uptake than on trabecular meshwork cells at concentrations ranging from 10(-1) to 10(-5) mg/ml after 3-, 5- and 60-min treatment, respectively. The living cells after mitomycin-C treatment were estimated with MTT assay that was converted tetrazolium dye of living cells only into insoluble purple formazan crystals within mitochondria. In the presence of mitomycin-C for 3, 5, and 60 min, the cellular MTT values in ciliary process cells were more decreased than in trabecular meshwork cells. Depolarization of the trabecular meshwork cells with 50 mM KCl led to an increase in intracellular calcium concentration, whereas application of mitomycin-C at 10(-3) mg/ml resulted in decrease of KCl-induced intracellular calcium increase. Mitomycin-C (10(-3) mg/ml) decreased cAMP concentration in ciliary process cells following 3- and 5-min treatment; however, it did not significantly affect the cellular cAMP concentration after only a 1-min exposure. Mitomycin-induced marked ladder pattern of DNA fragmentation was observed in ciliary process tissues after treatment with 10(-1) mg/ml of mitomycin-C for 3 and 5 min. However, the DNA pattern in trabecular meshwork tissues was not obviously affected by mitomycin-C. These findings from our results indicate that mitomycin-induced ocular hypotony may result from damage to both ciliary process and trabecular meshwork tissues.     

体外培养猴眼小梁细胞及睫状肌细胞中组织金属蛋白酶抑制剂的表达

目的：观察体外培养的正常猴眼小梁细胞及睫状肌细胞中组织金属蛋白酶抑制剂（tissue inhibitors of metalloproteinase,TIMP)的表达,探讨生理状态下基质金属蛋白酶（matrix metalloproteinase,MMP)及TIMP在小梁网房水流出及葡萄膜－巩膜房水流出的通道中的作用。方法：采用Reverse-zymography技术,检测猴眼小梁细胞及睫状肌细胞中TIMP及MMP的表达。结果;猴眼小梁细胞及睫状肌细胞培养液中均可见TMIP－1、TIMP－2及TIMP－3表达,小梁细胞中MMP／TIMP比值明显高于睫状肌细胞。结论：正常状态下小梁细胞外基质的降解能力明显高于睫状肌细胞,可能是由于传统的小梁网房水流出通道作用强于葡萄膜－巩膜房水流出通道 的作用。     

Immunofluorescence method for quantifying the trabecular meshwork glucocorticoid response (TIGR) protein in trabecular meshwork and Schlemm's canal cells.

PURPOSE. Decreased flow of aqueous humor through the trabecular meshwork (TM) and into Schlemm's canal (SC) is believed to be a predominant factor in the development of the elevated intraocular pressure found in primary open-angle and steroid glaucoma. Recent biochemical and genetic evidence has suggested that alterations in the expression of the TM glucocorticoid response (TIGR) protein within the chamber angle may play a role in the development of these glaucomas. To understand the process of TIGR induction in outflow pathway cells we developed an assay for TIGR expression that could distinguish both individual cell responses and also provide a semi-quantitative comparison between cell cultures. The present study demonstrates this approach, using digital image capture of immunofluorescently stained human TM and SC cells after treatment with dexamethasone (Dex). METHODS. Confluent cultures of human TM and SC cells were treated with 1 M Dex or vehicle control for 10 days. The cells were then fixed, permeabilized, and stained using polyclonal antibodies produced against recombinant TIGR. Digital images of fluorescently stained cells, using the same exposure time within an experiment, were evaluated by tabulating the staining intensity of all cells on each image. Between 10 and 40 cells were evaluated per image, 8-10 frames/ sample, 2-3 samples per treatment. Each cell was ranked as either 0 (background), 1+ (minor), 2+ (moderate) or 3+ (very bright staining). RESULTS. TM cells showed a significant basal level of TIGR staining. About 20% of control cells showed appreciable levels of TIGR staining, with intensity levels evenly distributed between 1, 2 and 3+. Dex treatment increased the number of TM cells expressing TIGR to 60-80%, with the majority of cells showing 2+ to 3+ staining throughout the cytoplasm. SC cells showed no basal TIGR staining, but Dex-treated cells exhibited TIGR staining in 6-15% of cells. SC cell TIGR staining varied between 1+ to 2+ in intensity, and showed a distribution different from TM cells. Staining in SC cells was localized to a ribbon-like compartment adjacent to the nucleus. Such perinuclear localization was rarely seen in TM cells. CONCLUSIONS. The low standard errors of the mean TIGR responses within each experiment, and the reproducibility between experiments for each cell type, suggests good reliability for this method. At the same time, the consistent, marked contrast between TM and SC cells in their response to glucocorticoids demonstrates that the assay can distinguish significant differences between cell types. The data support the view that Dex has a cell type specific effect on TIGR induction. The different extent, pattern and localization of TIGR staining between cell types suggests that TIGR expression in SC cells could play a functional role in the outflow pathway, but one that may be distinct from that played in TM cells.     

Cross-linked actin networks (CLANs) in bovine trabecular meshwork cells

A cytoskeletal feature of human trabecular meshwork (HTM) cells in vitro and ex vivo is the presence of cross-linked actin networks (CLANs) that are abundant in a proportion of TM cells exposed to dexamethasone (DEX) and also in cells from glaucoma patients. We wished to determine whether CLANs were present in the bovine trabecular meshwork (BTM), whether they were similarly induced by dexamethasone and whether the structures were comparable to CLANs in HTM cells. Cultures of HTM and BTM cells and ex vivo dissections of BTM tissue were stained with phalloidin (F-actin) and propidium iodide (nuclei) and imaged by confocal microscopy, thereafter being subjected to image analysis. Some CLAN-like structures were identified in ex vivo BTM tissue cultured with and without DEX. However we found that BTM cells in culture produced abundant CLANs when exposed to DEX; comparable to the best response from HTM cells. The CLANs were of similar dimensions and morphology to those found in human cells and they had a similar half life of 2 or 3 days following the removal of DEX. This work demonstrates that BTM cells provide a suitable model for future investigations of CLAN formation and function.BTM cultures are sufficiently hardy to thrive in low serum and serum-free conditions so we were able to show that aqueous humor stimulates CLAN formation in the target cells. Future research is directed at identifying the aqueous component(s) responsible for CLAN production.     

Junctions between the cells of the trabecular meshwork

Summary In an electron-microscopic study of cell-to-cell attachments in the trabecular meshwork of three primate species and the rabbit, two types of junctions were identified. These junctions were common to all four species. One was classified as a macula adhaerens while the other, after uranyl acetate en bloc treatment of the tissue, was thought to be a macular gap junction. These junctions were common to all regions of apposition in the meshwork although their incidence in isolated sections was low; their possible role as an adhesive mechanism was discussed.

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Zusammenfassung In einer elektronenmikroskopischen Studie der Intercellularverbindungen des Trabekelwerkes bei drei Primatenarten und beim Kaninchen wurden zwei Typen von Verbindungen identifiziert. Diese fanden sich gleichermaßen bei allen vier Species. Die eine Intercellularverbindung wurde als Macula adhaerens klassifiziert, während die andere nach en-bloc-Behandlung mit Uranylacetat für eine maculäre Gap junction gehalten wurde. Obwohl das Vorkommen dieser Verbindungen in Einzelschnitten relativ selten war, fanden sie sich doch überall dort, wo im Trabekelwerk Zellen aneinander stießen. Ihre mögliche Rolle im Sinne eines Adhäsionsmechanismus wird diskutiert.

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This paper was presented in part at the second annual meeting of the European Club for Ophthalmic Fine Structure in London on April 19 and 20, 1974.     

Human trabecular meshwork cells in culture: morphology and extracellular matrix components

This study demonstrates the presence of laminin and collagen type IV in the extracellular space of human trabecular meshwork (HTM) cells in culture and its absence in cultures of fibroblasts from sclera adjacent to the outflow pathway. These basement membrane components can be detected by standard immunohistochemical techniques. Positive staining for these macromolecules is found only after the HTM cells have reached confluence. The presence of laminin and human collagen type IV in the early passages can serve as additional criteria for identification of HTM cells in culture. These cells provide a useful experimental system for studying the effects of drugs and other factors on the synthesis of components of the extracellular matrix.     

睫状体和小梁网上相关离子通道的研究进展

房水由睫状上皮细胞产生,约80%通过小梁网进入Schlemm管,睫状上皮细胞和小梁网细胞上存在丰富的离子通道(Cl-通道、Ca~(2+)通道、K+通道和水通道蛋白等),这些通道对房水的产生和滤过有重要影响.本文对近年来睫状体和小梁网上的离子通道研究进行综述,希望能对青光眼研究和治疗有所帮助.     

小梁网干细胞的归巢及其在小梁创伤愈合中的作用

目的 研究胶原蛋白在小梁网干细胞（trabecular meshwork stem cells，TMSC）归巢中的作用以及小梁网（trabecular meshwork，TM）细胞和TMSC在创伤愈合中的相互作用。  设计 实验研究。研究对象 人原代TM细胞和TMSC。方法 分离、培养和传代人TM细胞和TMSC，通过定量RT-PCR和地塞米松诱导方法明确细胞类型；通过细胞贴附实验评价TM细胞及胶原蛋白对TMSC的亲和力；利用划痕损伤实验和时差显微镜观察TMSC分布及其与TM的相互作用。主要指标  TMSC和TM细胞的鉴定、TMSC穿膜数量和划痕损伤实验中TM细胞的迁移状态。 结果TMSC高表达OCT4而TM细胞高表达CHI3L1和MYOC，地塞米松刺激后TM细胞MYOC的表达显著增加。TMSC在含有TM细胞、胶原蛋白、TM细胞＋胶原蛋白的培养皿中的穿膜数分别为（13.1±5.3）个/高倍视野、（22.6±10.1）个/高倍视野、（4.8±2.2）个/高倍视野，均明显高于在单纯细胞培养皿中的穿膜数（3.7±0.5）个/高倍视野（P=0.0009，<0.0001，<0.001）。用AMD3100抑制TMSC中CXCR4的表达后，TMSC在各种状态的迁移数量差异消失。在TM细胞划痕损伤模型中加入TMSC可促进TM细胞的分化和迁移。结论  TMSC可诱导小梁网细胞分裂向受损区域迁移，从而促进组织损伤修复。胶原蛋白在TMSC归巢过程中发挥重要作用。     

Adenylyl cyclase activity mediated by beta-adrenoceptors in immortalized human trabecular meshwork and non-pigmented ciliary epithelial cells.

Non-pigmented ciliary epithelial (NPE) and trabecular meshwork (TM) cells are important in maintaining normal aqueous humor dynamics through the inflow and outflow routes, respectively. The current studies were undertaken to evaluate the ability of several beta-adrenergic receptor agonists to stimulate various antagonists to inhibit cAMP production in cultured immortalized human TM and NPE cells using an automated enzyme immunoassay. Isoproterenol was the most potent agonist in both the NPE and TM cells. The rank order of potency of agonists in NPE and TM cells, respectively, was: isoproterenol [EC50 = 37 and 66 nM] > epinephrine [EC50 = 112 and 526 nM] > albuterol [EC50 = 426 and 785 nM] > norepinephrine [EC50 = 3 and > 10 microM] > phenylephrine [EC50 > 10 microM for both] = dopamine [EC50 > 10 microM for both](n = 3-19). The isoproterenol-induced cAMP production was inhibited by various antagonists with the following rank order of potency in NPE and TM cells, respectively: propranolol [Ki = 0.2 and 0.3 nM] = ICI-118551 [Ki = 0.5 and 0.4 nM] > levobunolol [Ki = 1.1 and 2.1 nM] > levobetaxolol [Ki = 13 and 14 nM] = racemic betaxolol [Ki = 43 and 19 nM] > dextrobetaxolol [Ki = 2,705 and 1,980 nM] > atenolol [Ki > 4,000 nM for both] (n = 3-7). These detailed pharmacological studies using a variety of agonists and antagonists further supported the presence of beta2-adrenergic receptors in immortalized human NPE and TM cells.     

Cell-specific differential modulation of human trabecular meshwork cells by selective adenosine receptor agonists

Activation of A1 and A2A subtype adenosine receptors (AR) likely exert opposing effects on outflow of aqueous humor, and thereby, on intraocular pressure. Selective agonists of adenosine receptor (AR) subtypes have previously been applied to trabecular meshwork (TM) and Schlemm's canal (SC) cells to identify the site(s) of differential purinergic modulation. However, the apparent changes in volume monitored by previously measuring projected cell area might have partially reflected cell contraction and relaxation. In addition, whole-cell current responses of the TM cells previously described were highly variable following application of selective A1, A2A and A3 agonists. The complexity of the electrophysiologic responses may have reflected cell heterogeneity of the populations harvested from collagenase digestion of TM explants. We now report measurements of TM-cell volume using calcein fluorescence quenching, an approach independent of contractile state. Furthermore, we have applied selective AR agonists to a uniform population of human TM cells, the hTM5 cell line. A1, but not A2A or A3, AR agonists triggered TM-cell shrinkage. Both A1 and A2A AR agonists produced reproducible increases in TM-cell whole-cell currents of similar magnitude. The results suggest that previous measurements of explant-derived TM cells may have reflected a range of responses from phenotypically different cell populations, and that the opposing effects of A1 and A2A agonists on outflow resistance are not likely to be mediated by actions on a single population of TM cells. These opposing effects might reflect AR responses by two or more subpopulations of TM cells, by TM and SC cells or by inner-wall SC cells, alone.     

Adenosine A1 receptor modulation of MMP-2 secretion by trabecular meshwork cells

PURPOSE: Studies have shown that adenosine A(1) agonists can lower IOP in rabbits, mice, and monkeys, and this response is mediated in part by increases in outflow facility. The purpose of this project was to evaluate the response of trabecular meshwork cells to the addition of the adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA). METHODS: The human trabecular meshwork (HTM-3) cell line and primary cultures of bovine trabecular meshwork (BTM) cells were used in these studies. Cells were treated with CHA, and the secretion of matrix metalloproteinase (MMP)-2 or the activation of extracellular signal-regulated kinase (ERK1/2) was determined. RESULTS: Treatment of HTM-3 and BTM cells with CHA (0.1 micro M) resulted in a time-dependent secretion of MMP-2 that was measurable as early as 30 minutes after treatment and reached a maximum by 2 hours. This CHA-induced secretion of MMP-2 was inhibited by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) and by the ERK1/2 pathway inhibitor U0126. Treatment of HTM-3 cells with CHA produced a rapid dose-dependent activation of ERK1/2 with an EC(50) of 5.7 nM. The CHA-induced activation of ERK1/2 was inhibited by pretreatment with the adenosine A(1) antagonist CPT and by the ERK pathway inhibitor U0126. CONCLUSIONS: The addition of the adenosine A(1) agonist CHA stimulates the secretion of MMP-2 from trabecular meshwork cells. This secretory response involves the activation of adenosine A(1)-linked stimulation of ERK1/2. These results provide evidence for the existence of functional adenosine A(1) receptors in the trabecular cells and that the activation of these receptors stimulates secretion of MMP-2.