Hsp70 protects human trabecular meshwork cells injury induced by UVB through Smad pathway.

AIM: Glaucoma is a universal eye disease which is commonly associated with increased intraocular pressure caused by impaired aqueous humor (AH) drainage. This study aimed to investigate the effects of Hsp70 on trabecular meshwork (TM) injury induced by UVB. METHODS: Real-time quantitative PCR (qRT-PCR) was used to examine the mRNA levels of Hsp70. siRNA was used to downregulate Hsp70 expression in the TM cells to inspect changes in cell proliferation and apoptosis. Cell proliferation was assessed by a Cell Counting Kit-8 (CCK-8) assay and the number of apoptotic cells was assessed using annexin V-FITC/PI apoptosis detection kit. The Smad signaling pathway was investigated using western blotting analyses. RESULTS: The overexpression of Hsp70 promoted cell proliferation and suppressed apoptosis. What's more, the overexpression of Hsp70 suppressed the expression of Smad-2, Smad-3 and Smad-7. CONCLUSION: Hsp70 might improve cell viability and inhibit TM apoptosis by inhibition of the Smad pathway. Hsp70 is a potential therapeutic target for the treatment of glaucoma.

miR-183 Inhibits UV-Induced DNA Damage Repair in Human Trabecular Meshwork Cells by Targeting of KIAA0101.

PURPOSE: The purpose of this study was to investigate the mechanisms by which miR-183 may contribute to the phenotypic alterations associated with stress-induced senescence of human trabecular meshwork (HTM) cells. METHODS: Changes in gene expression induced by miR-183 in HTM cells were evaluated by gene array analysis, confirmed by quantitative-PCR (Q-PCR), and analyzed by MetaCore pathway analysis. Effects of miR-183 on cell proliferation were assessed by incorporation of bromodeoxyuridine incorporation, and DNA damage by CometAssay after ultraviolet (UV) irradiation in primary HTM cells, and confirmed in human diploid fibroblasts (HDF) and HeLa cells. A plasmid expressing KIAA0101 without its 3'-untranslated region (3'-UTR) was cotransfected with miR-183 to evaluate the role of KIAA0101 on the effects induced by miR-183. RESULTS: miR-183 affected the expression of multiple genes involved in cell cycle regulation and DNA damage response in HTM cells. Forced expression of miR-183 in HTM and HDF resulted in a significant decrease in proliferation in primary HTM and HDF cells but not in HeLa cells. In all cell types tested, overexpression of miR-183 resulted in increased DNA damage under UV irradiation. Expression of KIAA0101 lacking the 3'-UTR region partially prevented the effects of miR-183 on cell proliferation and completely reversed the effects on UV-induced DNA damage. CONCLUSIONS: Our results suggest that the observed up-regulation of miR-183 after stress-induced senescence in HTM cells may contribute to reinforce cellular senescence by inhibiting cell cycle progression through multiple gene targets and limiting the DNA repair mechanisms through inhibition of KIAA0101.

Profiling of Cytokines Secreted by Conventional Aqueous Outflow Pathway Endothelial Cells Activated In Vitro and Ex Vivo With Laser Irradiation.

PURPOSE: To profile which cytokine genes are differentially expressed (DE) as up- or downregulated by cultured human trabecular meshwork (TMEs) and Schlemm's canal endothelial cells (SCEs) after three experimental treatments consisting of selective laser trabeculoplasty (SLT) irradiation, exposure to media conditioned either by SLT-irradiated TMEs (TME-cm) or by SCEs (SCE-cm). Also, to profile which cytokines are upregulated ex vivo in SLT-irradiated human conventional aqueous outflow pathway (CAOP) tissues. METHODS: After each treatment, Affymetrix microarray assays were used to detect upregulated and downregulated genes for cytokines and their receptors in TMEs and SCEs. ELISA and protein antibody arrays were used to detect upregulated cytokines secreted in SLT-irradiated CAOP tissues ex vivo. RESULTS: The SLT irradiation upregulated numerous cytokine genes in TMEs, but only a few in SCEs. Exposure to TME- and SCE-cm induced SCEs to upregulate many more cytokine genes than TMEs. Selective laser trabeculoplasty irradiation and exposure to TME-cm downregulated several cytokine genes in TMEs but none in SCEs. Selective laser trabeculoplasty irradiation induced one upregulated and three downregulated cytokine-receptor genes in TMEs but none in SCEs. Exposure to TME-cm induced upregulation of one and downregulation of another receptor gene in TMEs, whereas two unique cytokine-receptor genes were upregulated in SCEs. Cytokine protein expression analysis showed that at least eight cytokines were upregulated in SLT-irradiated human CAOP tissues in situ/ex vivo. CONCLUSIONS: This study has helped us identify a cytokine signaling pathway and to consider newly identified mechanisms regulating aqueous outflow that may lay the foundation for the future development of cytokine-based glaucoma therapies.

A laser-induced mouse model with long-term intraocular pressure elevation.

PURPOSE: To develop and characterize a mouse model with intraocular pressure (IOP) elevation after laser photocoagulation on the trabecular meshwork (TM), which may serve as a model to investigate the potential of stem cell-based therapies for glaucoma. METHODS: IOP was measured in 281 adult C57BL/6 mice to determine normal IOP range. IOP elevation was induced unilaterally in 50 adult mice, by targeting the TM through the limbus with a 532-nm diode laser. IOP was measured up to 24 weeks post-treatment. The optic nerve damage was detected by electroretinography and assessed by semiautomatic counting of optic nerve axons. Effects of laser treatment on the TM were evaluated by histology, immunofluorescence staining, optical coherence tomography (OCT) and transmission electron microscopy (TEM). RESULTS: The average IOP of C57BL/6 mice was 14.5 ± 2.6 mmHg (Mean ± SD). After laser treatment, IOP averaged above 20 mmHg throughout the follow-up period of 24 weeks. At 24 weeks, 57% of treated eyes had elevated IOP with the mean IOP of 22.5 ± 2.5 mmHg (Mean ± SED). The difference of average axon count (59.0%) between laser treated and untreated eyes was statistically significant. Photopic negative response (PhNR) by electroretinography was significantly decreased. CD45+ inflammatory cells invaded the TM within 1 week. The expression of SPARC was increased in the TM from 1 to 12 weeks. Histology showed the anterior chamber angle open after laser treatment. OCT indicated that most of the eyes with laser treatment had no synechia in the anterior chamber angles. TEM demonstrated disorganized and compacted extracellular matrix in the TM. CONCLUSIONS: An experimental murine ocular hypertension model with an open angle and optic nerve axon loss was produced with laser photocoagulation, which could be used to investigate stem cell-based therapies for restoration of the outflow pathway integrity for ocular hypertension or glaucoma.

Cellular processing of myocilin.

BACKGROUND: Myocilin (MYOC) is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Pro370Leu (P370L) and Gln368stop (Q368X) have been identified in patients. In the present study, we investigated the processing of myocilin in human trabecular meshwork (TM) cells as well as in inducible, stable RGC5 cell lines. METHODOLOGY/PRINCIPAL FINDINGS: The turnover and photoactivation experiments revealed that the endogenous myocilin in human trabecular meshwork (TM) cells was a short-lived protein. It was found that the endogenous myocilin level in TM cells was increased by treatment of lysosomal and proteasomal inhibitors, but not by autophagic inhibitor. Multiple bands immunoreactive to anti-ubiquitin were seen in the myocilin pull down, indicating that myocilin was ubiquitinated. In inducible cell lines, the turnover rate of overexpressed wild-type and mutant P370L and Q368X myocilin-GFP fusion proteins was much prolonged. The proteasome function was compromised and autophagy was induced. A decreased PSMB5 level and an increased level of autophagic marker, LC3, were demonstrated. CONCLUSIONS/SIGNIFICANCE: The current study provided evidence that in normal homeostatic situation, the turnover of endogenous myocilin involves ubiquitin-proteasome and lysosomal pathways. When myocilin was upregulated or mutated, the ubiquitin-proteasome function is compromised and autophagy is induced. Knowledge of the degradation pathways acting on myocilin can help in design of novel therapeutic strategies for myocilin-related glaucoma.

Effect of low fluence diode laser irradiation on the hydraulic conductivity of perfused trabecular meshwork endothelial cell monolayers.

OBJECTIVE: To determine the effect of low-fluence diode laser irradiation upon the fluid perfusion characteristics of cultured human trabecular meshwork cell monolayers when placed in a specially designed testing apparatus and subjected to fluid flow driven by a hydrostatic pressure gradient. METHODS: Two experimental series were conducted. In the first series, six low-fluence diode laser irradiation experiments were conducted using cultured human trabecular meshwork cell monolayers grown on filter supports. Upon reaching a steady state perfusion condition at approximately 5.0 mmHg, monolayers were irradiated at fluencies ranging from 0.2619 to 0.8571 J/cm2 using a diode laser (lambda=810 nm). Perfusion and data collection continued for 45 minutes post-irradiation, after which the monolayers were tested to determine post-experimental viability. Hydraulic conductivity values were analyzed for post-irradiation response in 2.5-minute intervals, grouped by viability. In the second series, a total of six irradiated experiments and six simultaneous nonirradiated control experiments were conducted. Fluence values of 0.3571 J/cm2 (n=3) and 0.4286 J/cm2 (n=3) were used. Hydraulic conductivity values were analyzed for post-irradiation response in 2.5-minute intervals, grouped by irradiated vs. nonirradiated control groups. RESULTS: In the first series, analysis showed that the viable monolayers exhibited a statistically significant increase in hydraulic conductivity (p<0.001) from 10 minutes post-irradiation onward. The non-viable monolayers exhibited a statistically significant decrease in hydraulic conductivity. In the second series, irradiated groups showed a significant difference (p<0.001) from nonirradiated controls from 10 minutes post-irradiation onward. CONCLUSION: Low-fluence diode laser irradiation increases hydraulic conductivity in viable perfused TM cell monolayers when compared to baseline values or simultaneous nonirradiated controls while decreasing hydraulic conductivity in nonviable monolayers.

A new insight into the cellular regulation of aqueous outflow: how trabecular meshwork endothelial cells drive a mechanism that regulates the permeability of Schlemm's canal endothelial cells.

AIM: To test the hypothesis that trabecular meshwork endothelial cells (TMEs) increase the permeability of Schlemm's canal endothelial cells (SCEs) by actively releasing ligands that modulate the barrier properties of SCEs. METHODS: The TMEs were first irradiated with a laser light and allowed to condition the medium, which is then added to SCEs. The treatment response is determined by both measuring SCE permeability (flow meters) and the differential expression of genes (Affymetrix chips and quantitative polymerase chain reaction (PCR)). The cytokines secreted by the treated cells were identified using ELISA and the ability of these cytokines to increase permeability is tested directly after their addition to SCEs in perfusion experiments. RESULTS: SCEs exposed to medium conditioned by the light activated TMEs (TME-cm) respond by undergoing a differential expression (DE) of 1,120 genes relative to controls. This response is intense relative to a DE of only 12 genes in lasered SCEs. The TME-cm treatment of SCEs increased the SCE permeability fourfold. The role of cytokines in these responses is supported by two findings: adding specific cytokines established to be secreted by lasered TMEs to SCEs increases permeability; and inactivating the TME-cm by boiling or diluting, abrogates these conditioned media permeability effects. CONCLUSION: These experiments show that TMEs can regulate SCE permeability and that it is likely that TMEs have a major role in the regulation of aqueous outflow. This novel TME driven cellular mechanism has important implications for the pathogenesis of glaucoma and the mechanism of action of laser trabeculoplasty. Ligands identified as regulating SCE permeability have potential use for glaucoma therapy.

Interactions between endothelia of the trabecular meshwork and of Schlemm's canal: a new insight into the regulation of aqueous outflow in the eye.

PURPOSE: To test the hypothesis that trabecular meshwork endothelial cells (TMEs) regulate aqueous outflow by actively releasing ligands that upon binding to Schlemm's canal endothelial cells (SCEs) increase transendothelial flow, thereby facilitating the egress of aqueous. METHODS: We tested our hypothesis by (1) activating the TMEs in vitro using a laser procedure known to increase aqueous outflow in vivo; (2) demonstrating that lasered TMEs become activated at the genome-wide level and synthesize ligands; (3) ascertaining that media conditioned by laser-activated TMEs and ligands therein increase transendothelial flow when added to SCEs; and (4) determining that ligands identified as synthesized by TMEs increase permeability when added to SCEs. RESULTS: We find that adding either media conditioned by lasered TMEs or ligands synthesized by TMEs to naïve control SCEs increases permeability. Adding media boiled, diluted, or conditioned by nonlasered TMEs abrogates these permeability effects. Media conditioned by either lasered TMEs or SCEs (TME-cm/SCE-cm), when added to untreated controls of each cell type, induce congruous gene expression and flow effects: TME-cm induces far more differentially expressed genes (829 in control TMEs and 1,120 in control SCEs) than does the SCE-cm (12 in control TMEs and 328 in control SCEs), and TME-cm also increases flow much more (more than 11-fold in control TMEs and more than fourfold in control SCEs) than does the SCE-cm (fivefold in control TMEs and twofold in control SCEs). CONCLUSIONS: As postulated, the TMEs release factors that regulate SCE permeability. Derangement of this TME-driven process may play an important role in the pathogenesis of glaucoma. Ligands identified, which regulate permeability, have potential use for glaucoma therapy.

Effects of visible light on cultured bovine trabecular cells.

To explore the biological effects of light on trabecular cells, cultured bovine trabecular cells were exposed to visible light of different wavelength with different energy. Cellular morphology, structure, proliferation, and phagocytosis were observed. The cells showed no remarkable changes when the energy was low. When the exposure energy reached 1.12 mW/cm2, the cytoplasm showed a rough appearance, and cell proliferation and phagocytosis decreased. This phototoxicity was strong with white light (compound chromatic light), moderate with violet light or yellow light, and mild with red light.

Trabeculectomy with beta radiation: long-term follow-up.

PURPOSE: To evaluate the long-term outcome and complications of trabeculectomy with beta radiation. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: Forty-three patients with confirmed primary open-angle glaucoma (POAG), who received trabeculectomy with adjunctive beta radiation at the Prince of Wales Hospital between June 1991 and November 1994. METHODS: Patients fulfilling the preceding criteria were followed up longitudinally. The visual acuity, intraocular pressure (IOP), bleb morphology, and complications were evaluated. MAIN OUTCOME MEASURES: Visual acuity, IOP, bleb morphology, complications. RESULTS: Forty-three eyes of 43 consecutive Chinese patients were recruited and successfully followed up for a minimum of 7 years. The mean age +/- 1 standard deviation (SD) was 60.9 +/- 13.0 years. There were 29 males and 14 females. The mean baseline IOP +/- 1 SD was 28.3 +/- 5.8 mmHg. The mean postoperative IOP +/- 1 SD after the initial trabeculectomy was 11.9 +/- 4.3 mmHg, and the mean number of preoperative IOP-lowering eyedrops +/- 1 SD was reduced from 2.8 +/- 0.5 to 0.7 +/- 1.0 at 7 years follow-up. The qualified success rate at 7 years follow-up, defined as IOP

Acute ultrastructural changes of the trabecular meshwork after selective laser trabeculoplasty and low power argon laser trabeculoplasty.

BACKGROUND AND OBJECTIVES: To compare the histopathological changes in the human trabecular meshwork after low power argon laser trabeculoplasty (ALT) and selective laser trabeculoplasty (SLT) with a Q-switched, frequency-doubled, neodymium:yttrium-aluminium-garnet (Nd:YAG) laser. STUDY DESIGN/MATERIALS AND METHODS: In gonioscopically normal trabecular meshwork of three patients awaiting enucleation due to malignant melanoma of the choroid, SLT and ALT were performed 1-5 days prior to enucleation. In each eye, the lower half of trabecular meshwork received SLT, one quadrant low power (460 mW) ALT and one quadrant was left untreated. Specimens were evaluated with light and transmission electron microscopy. RESULTS: A sharp demarcation line was visible between the laser treated and untreated intact trabecular meshwork after ALT and SLT. Both lasers caused disruption of trabecular beams, but the extent of the damage was smaller after SLT. The collagen component of trabecular beams was mostly amorphous, the long-spacing collagen was scanty after ALT, but more abundant after SLT. In the intertrabecular spaces fragmented cells and tissue debris with only a few pigmented cells were observed. Some endothelial cells were desquamated, but appeared slightly better preserved after SLT than ALT. CONCLUSIONS: Our ultrastructural comparison of the morphological changes after low power ALT and SLT in patients demonstrated that both lasers caused splitting and fragmentation of the trabecular beams of the trabecular meshwork, but the extent of the damage was smaller and the preservation of long-spacing collagen better after SLT than after ALT.

Clinical, structural and molecular phototherapy effects of laser irradiation on the trabecular meshwork of human glaucomatous eyes.

The effects of argon laser trabeculoplasty (LTP) on intraocular pressure (IOP), outflow facility, the morphology of the trabecular meshwork (TM), and the pattern of extracellular glycoprotein fibronectin in trabeculum were studied in 46 eyes of patients with primary open-angle glaucoma (POAG). The LTP was done with informed consent, anticipating that trabeculectomy would be carried out at a scheduled time (2 h to several months following laser therapy). We found that the magnitude of IOP reduction and the improvement in the facility of outflow achieved are directly dependent on the time course after LTP and laser-induced structural changes in trabecular tissue. Light microscopic and immunohistochemical evaluations of the TM specimens at earlier intervals after LTP revealed evidence of heat effects, with disruption and shrinkage of the TM collagenous components and accumulation of fibronectin deposits in the aqueous drainage channels as compared with the TMs of matched patients with POAG who did not receive laser treatment. Within 24 h after LTP, proteins of glaucomatous TMs excised from patients incorporated increased amounts of [3H]-leucine radioactive label; however, the amount of [3H]-leucine-labeled material was significantly depressed in later periods of evaluation. The specimens obtained at longer intervals after LTP showed partial or total occlusion of the intertrabecular spaces by extracellular debris; however, the amount of trabecular fibronectin was not significantly different from that measured 24 h after LTP. At least two potential mechanisms are proposed for the TM tissue response to laser treatment, including heat-induced stretching of the collagen in lamellae and fibronectin-mediated attachment of beams supporting an adhesive tightening of the trabecular components caused by LTP. The changes in laser-induced tissue responses appear to be the result of morphological repair of irradiation-injured trabecular tissue.

Stimulation of cell division by argon and Nd:YAG laser trabeculoplasty in cynomolgus monkeys.

Although laser treatment of the trabecular meshwork is the most common form of surgery for glaucoma, the tissue response to this therapy is still incompletely understood. We applied argon or Nd:YAG laser to the trabecular meshwork of six monkeys. Cell division was identified by injecting tritiated thymidine into the anterior chamber 24 hr after laser application. Autoradiography of tissue sections revealed significantly more labelled cells in eyes treated with laser than in the untreated controls. In addition, cells in neighboring tissues such as iris, ciliary body and sclera showed labelling in association with laser application. Furthermore, comparison of argon-induced lesions with those caused by pulsed Nd:YAG suggests that there are quantitative and qualitative differences in the response of trabecular meshwork and surrounding tissues to these two forms of laser energy.

Morphology of the trabecular meshwork within monkey (Macaca speciosa) eyes after irradiation with the free-running Nd:YAG laser.

The long-term tissue response of monkey eyes to trabeculoplasty produced at the chamber angle by neodymium:YAG (Nd:YAG) laser (thermal mode) was investigated. Four weeks after irradiation, a monolayer of corneal-like endothelial cells extended over the trabeculum in impact zones, with partial occlusion of intertrabecular spaces; this condition persisted at all subsequent stages of observation (viz, up to 13 weeks). Both primary and secondary degeneration of trabecular endothelial cells occurred predominantly at the posterior border of the juxtacanalicular tissue, and continued throughout the observation period. Lysis and degradation of collagen fibrils were also prominent in this region, and collagen-phagocytosing cells could be seen at all stages. Although activated fibroblasts were marked in this zone, regeneration processes were, in general, less marked than those of degeneration. The significance of these findings in relation to the mechanism by which trabeculoplasty works is discussed.

The effect of argon laser trabeculoplasty on the rate of filtering surgery.

We examined the impact of argon laser trabeculoplasty (ALT) on the overall rate of filtering surgery on a resident service. From 1981 through 1984, 121 eyes underwent ALT for treatment of uncontrolled primary open-angle glaucoma despite maximum tolerated medical therapy. Mean intraocular pressure decreased from 27 +/- 4 mmHg to 20 +/- 5 mmHg (median follow-up, 9 months). From 1978 through 1981, the mean annual rate of filtering surgery was 16. Only one filtering procedure was performed in 1982, the first full year of ALT use. However, 15 filtering procedures were performed in 1983 and 18 were performed in 1984. Despite a large number of apparently effective ALT treatments and despite an overall reduction in outpatient visits of 20%, the rate of filtering surgery has returned to the pre-ALT level. This suggests that ALT may be effective in delaying the need for surgery, but in many cases it probably does not prevent surgery.

Thermal effects on the trabecular meshwork induced by laser irradiation: clinical implications deduced from ultrastructural studies on the macaca speciosa monkey.

The pressure lowering effect brought about by irradiating the chamber angle with argon laser light according to Wise and Witter was studied by ultrastructural methods and was analysed in order to ascertain the mechanism responsible. To this end, the effects induced in 4 macaca speciosa monkeys by such irradiation were examined using scanning and transmission electron microscopy. It was noted that the phenomena observed depend critically on the precise location of the irradiation site. When the anterior trabecular meshwork is irradiated, a proliferation of monocellular, atypical corneal endothelium cells occurs which tightly surround the irradiation location. In irradiation of the posterior trabecular meshwork, however, this effect is much less prominent. In both cases, by the end of the twelfth week a progressive, lytic degeneration of the entire trabecular meshwork was observed which reached the inner wall of Schlemm's canal.

Short-term effect of argon laser trabeculoplasty in monkeys.

We performed argon laser trabeculoplasty (ALT) in four cynomolgus monkeys and evaluated the morphologic changes in the trabecular meshwork one hour and 14 hours following the laser treatment. One hour after ALT, disruption of the trabecular beams and coagulative necrosis of the tissue were evident. Fragmented cells and fibrillar tissue debris were found in the trabecular spaces, accumulating in the juxtacanalicular region. This might explain the elevation of intraocular pressure seen immediately following ALT in some glaucomatous patients. Also, many trabecular endothelial cells were rounded up, displaying different stages of leaving the beams; these cells were actively phagocytic. Such stimulation of trabecular meshwork cells with subsequent removal of tissue debris might explain the temporary nature of elevation of intraocular pressure following ALT.

Laser therapy for glaucoma.

Three laser techniques for the treatment of glaucoma are described. The short-burn technique of argon laser iridotomy permitted perforation of the peripheral iris in one session without serious complications, thereby alleviating pupillary block in angle-closure glaucoma. The rates of successful intraocular pressure (IOP) control and visual acuity loss due to cataract progression were similar in laser iridotomy and surgical iridectomy. The tonographic outflow facility remained unchanged after argon laser iridotomy. Techniques of argon laser trabeculoplasty (ALT) to the anterior and posterior aspects of the trabecular band were evaluated. The anterior ALT gave fewer complications, but a lower IOP control rate than the posterior ALT. Posterior ALT over 180 degrees with about 50 burns gave fewer complications than treatment over 360 degrees with about 100 burns, yet the final effects in IOP control were better after the 180 degrees than after the 360 degrees treatment. Nd-YAG laser irradiation can separate dysgenetic iris insertion from the trabecular band in developmental glaucoma without serious complications. This Nd-YAG laser goniotomy may be useful for the treatment of juvenile primary developmental glaucoma.