Ferroptosis in Glaucoma: A Promising Avenue for Therapy.

Glaucoma, a blind-leading disease largely since chronic pathological intraocular high pressure (ph-IOP). Hitherto, it is reckoned incurable for irreversible neural damage and challenges in managing IOP. Thus, it is significant to develop neuroprotective strategies. Ferroptosis, initially identified as an iron-dependent regulated death that triggers Fenton reactions and culminates in lipid peroxidation (LPO), has emerged as a focal point in multiple tumors and neurodegenerative diseases. Researches show that iron homeostasis play critical roles in the optic nerve (ON) and retinal ganglion cells (RGCs), suggesting targeted treatments could be effective. In glaucoma, apart from neural lesions, disrupted metal balance and increased oxidative stress in trabecular meshwork (TM) are observed. These disturbances lead to extracellular matrix excretion disorders, known as sclerotic mechanisms, resulting in refractory blockages. Importantly, oxidative stress, a significant downstream effect of ferroptosis, is also a key factor in cell senescence. It plays a crucial role in both the etiology and risk of glaucoma. Moreover, ferroptosis also induces non-infectious inflammation, which exacerbate glaucomatous injury. Therefore, the relevance of ferroptosis in glaucoma is extensive and multifaceted. In this review, the study delves into the current understanding of ferroptosis mechanisms in glaucoma, aiming to provide clues to inform clinical therapeutic practices.

Regulatory role of cholesterol in modulating actin dynamics and cell adhesive interactions in the trabecular meshwork.

The trabecular meshwork (TM) tissue plays a crucial role in maintaining intraocular pressure (IOP) homeostasis. Increased TM contractility and stiffness are directly correlated with elevated IOP. Although cholesterol is known to be a determinant of glaucoma occurrence and elevated IOP, the underlying mechanisms remain elusive. In this study, we used human TM (HTM) cells to unravel the effects of cholesterol on TM stiffness. We achieved this by performing acute cholesterol depletion with Methyl-ß-cyclodextrin (MßCD) and cholesterol enrichment/replenishment with MßCD cholesterol complex (CHOL). Interestingly, cholesterol depletion triggered notable actin depolymerization and decreased focal adhesion formation, while enrichment/replenishment promoted actin polymerization, requiring the presence of actin monomers. Using a specific reporter of phosphatidylinositol 4,5-bisphosphate (PIP2), we demonstrated that cholesterol depletion decreases PIP2 levels on the cell membrane, whereas enrichment increases them. Given the critical role of PIP2 in actin remodeling and focal adhesion formation, we postulate that cholesterol regulates actin dynamics by modulating PIP2 levels on the membrane. Furthermore, we showed that cholesterol levels regulate integrin α5ß1 and αVß3 distribution and activation, subsequently altering cell-extracellular matrix (ECM) interactions. Notably, the depletion of cholesterol, as a major lipid constituent of the cell membrane, led to a decrease in HTM cell membrane tension, which was reversed upon cholesterol replenishment. Overall, our systematic exploration of cholesterol modulation on TM stiffness highlights the critical importance of maintaining appropriate membrane and cellular cholesterol levels for achieving IOP homeostasis.

Anti-apoptotic and autophagic effect: Using conditioned medium from human bone marrow mesenchymal stem cells to treat human trabecular meshwork cells.

Introduction: Glaucoma is a vision-threatening disease associated with accelerated aging of trabecular meshwork (TM) which results in elevated intraocular pressure (IOP). Increased oxidative stress in TM plays an important role in cellular molecular damage which leads to senescence. Autophagy is an intracellular lysosomal degradation process which is activated when cells are under stressful condition, and emerging studies have demonstrated increased expression of modulators of apoptosis and expression of autophagic cascade in ex-vivo TM specimens or cultured TM cells under oxidative stress. Recently, studies have shown neuroprotective and IOP-lowering effects after transplanting mesenchymal stem cells (MSCs) or injecting condition medium (CM) of MSCs into ocular hypertension animal models. However, knowledge of the underlying mechanism accounting for these effects is limited. Using condition medium (CM) from human bone marrow-derived mesenchymal stem cells (BM-MSCs), we investigated the effects of the CM derived from BM-MSCs on TM autophagy and apoptosis. Methods: H2O2 was added to culture medium of human TM cells to mimic oxidative damage in glaucomatous eyes, and the autophagic and anti-apoptotic effects of BM-MSCs-derived CM was explored on the oxidatively damaged cells. Mitochondrial ROS production was examined by MitoSOX™, apoptosis was evaluated using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) staining, and the expression of proteins involved in autophagy as well as extracellular matrix was investigated via Western blot. Results: There were no significant differences in TM cell viability when the cells were treated with different concentrations of CM in the absence of oxidative stress. Cell viability was significantly higher in oxidatively damaged TM cells treated with 1X or 5X CM compared to untreated TM cells under oxidative stress. The mitochondrial ROS level significantly increased with oxidative stress, which was mitigated in the CM treatment groups. DNA fragmentation significantly decreased in oxidatively stressed TM cells after treatment with CM. LCB3 II/LCB3 I was significantly elevated in the oxidative stress group compared to the control group and was significantly decreased in the CM treatment groups. Expression of fibronectin was not significantly different among the groups. Conclusion: The CM derived from human BM-MSCs has the capacity to rescue oxidatively damaged human TM cells associated with decreased autophagy and apoptosis. The BM-MSCs CM has potential for slowing down age- and disease-related degeneration of TM in patients with glaucoma, facilitating success in the control of IOP.

Analysis of Lipid Contents in Human Trabecular Meshwork Cells by Multiple Reaction Monitoring (MRM) Profiling Lipidomics.

Lipids are among the major constituents of cells and play many important cellular functions. Lipid levels in the trabecular meshwork (TM) aqueous humor outflow pathway play an important role in the maintenance of aqueous humor drainage and intraocular pressure (IOP) homeostasis. Therefore, it is important to characterize the changes in the lipid contents in the aqueous humor outflow pathway tissues to better understand their functional significance in the maintenance of IOP. The multiple reaction monitoring (MRM)-based profiling aids in the analysis of the metabolome as a collection of functional groups and is utilized as an exploratory metabolomics and lipidomics approach. The MRM-based profiling utilizes tandem mass spectrometry experiments carried out on a commercial triple quadrupole mass spectrometer with three aligned quadrupole mass filters (Q1, Q2, and Q3). This screening methodology can be utilized for targeted lipidomics screening. This chapter focuses on the methodology for isolation and culturing of the TM cells, lipid extraction, and the MRM-based lipidomics approach with data analysis.

Cell senescence alters responses of porcine trabecular meshwork cells to shear stress.

Mechanical microenvironment and cellular senescence of trabecular meshwork cells (TMCs) are suspected to play a vital role in primary open-angle glaucoma pathogenesis. However, central questions remain about the effect of shear stress on TMCs and how aging affects this process. We have investigated the effect of shear stress on the biomechanical properties and extracellular matrix regulation of normal and senescent TMCs. We found a more significant promotion of Fctin formation, a more obvious realignment of F-actin fibers, and a more remarkable increase in the stiffness of normal cells in response to the shear stress, in comparison with that of senescent cells. Further, as compared to normal cells, senescent cells show a reduced extracellular matrix turnover after shear stress stimulation, which might be attributed to the different phosphorylation levels of the extracellular signal-regulated kinase. Our results suggest that TMCs are able to sense and respond to the shear stress and cellular senescence undermines the mechanobiological response, which may lead to progressive failure of cellular TM function with age.

Airborne particulate matter (PM2.5) triggers ocular hypertension and glaucoma through pyroptosis.

BACKGROUND: Particulate matter (PM) is strongly linked to human health and has detrimental effects on the eye. Studies have, however, focused on the ocular surface, with limited research on the impact of PM2.5 on intraocular pressure (IOP). METHODS: To investigate the impact of PM2.5 on IOP and the associated mechanism, C57BL/6 mouse eyes were topically exposed to a PM2.5 suspension for 3 months, and human trabecular meshwork (HTM) cells were subjected to various PM2.5 concentrations in vitro. Cell viability, NLRP3/caspase-1, IL-1ß, and GSDMD expression, reactive oxygen species (ROS) production and cell contractility were measured by western blot, ELISA, cell counting kit-8, ROS assay kit or a cell contractility assay. ROS scavenger N-acetyl-L-cysteine (NAC) and caspase-1 inhibitor VX-765 were used to intervene in PM2.5-induced damages. RESULTS: The results revealed that the IOP increased gradually after PM2.5 exposure, and upregulations of the NLRP3 inflammasome, caspase-1, IL-1ß, and GSDMD protein levels were observed in outflow tissues. PM2.5 exposure decreased HTM cell viability and affected contraction. Furthermore, elevated ROS levels were observed as well as an activation of the NLRP3 inflammasome and downstream inflammatory factors caspase-1 and IL-1ß. NAC improved HTM cell viability, inhibited the activation of the NLRP3 inflammasome axis, and HTM cell contraction by scavenging ROS. VX-765 showed similar protection against the PM2.5 induced adverse effects. CONCLUSION: This study provides novel evidence that PM2.5 has a direct toxic effect on intraocular tissues and may contribute to the initiation and development of ocular hypertension and glaucoma. This occurs as a result of increased oxidative stress and the subsequent induction of NLRP3 inflammasome mediated pyroptosis in trabecular meshwork cells.

Interleukin-6-mediated trans-signaling inhibits transforming growth factor-ß signaling in trabecular meshwork cells.

Glaucoma is one of the major causes of blindness, and transforming growth factor-ß2 (TGF-ß2) has been found to be elevated in the aqueous humor of eyes with primary open-angle glaucoma (POAG). TGF-ß2 in aqueous humor causes the glaucoma-related fibrosis of human trabecular meshwork (HTM), suggesting an important role of TGF-ß in POAG pathogenesis. Here, we sought to elucidate the effects of IL-6 trans-signaling on TGF-ß signaling in HTM cells. Using a multiplex immunoassay, POAG patients decreased IL-6 levels and increased soluble IL-6 receptor (sIL-6R) levels compared with the controls. In in vitro experiments, we observed that the IL-6 level was increased in the conditioned medium of HTM cells after TGF-ß2 stimulation. To elucidate the relationship between TGF-ß2 and IL-6 in HTM cells, we conducted Western blotting and immunohistochemical analyses, and we noted that the combination of IL-6 and sIL-6R (IL6/sIL-6R) suppressed TGF-ß-induced up-regulation of α-smooth muscle actin in HTM cells, whereas IL-6 alone did not. This suggests that trans-signaling, not classic signaling, of IL-6 suppresses TGF-ß-induced fibrosis of HTM. IL6/sIL-6R also suppressed TGF-ß-mediated activation of myosin light chain 2 (MLC2), Smad2, and p38. Of note, these inhibitory effects of IL6/sIL-6R on TGF-ß were partly reduced by siRNA-mediated knockdown of STAT3. Moreover, IL-6/sIL-6R partly inhibited TGF-ß-induced activation of the Smad-sensitive promoter detected with luciferase reporter gene assays and up-regulation of TGFRI and TGFRII, evaluated by quantitative real-time RT-PCR. Strikingly, overexpression of TGFRI and TGFRII diminished these inhibitory effects of IL-6/sIL-6R. We conclude that of IL-6-mediated trans-signaling potently represses TGF-ß signaling in HTM cells.

Small-molecule inhibition of Wnt signaling abrogates dexamethasone-induced phenotype of primary human trabecular meshwork cells.

Trabecular meshwork (TM) cells are the governing regulators of the TM structure. When the functionality of these cells is impaired, the structure of the TM is perturbed which often results in increased ocular hypertension. High intraocular pressure is the most significant risk factor for steroid-induced glaucoma. Dexamethasone (Dex)-induced phenotype of TM cells is widely utilized as a model system to gain insight into mechanisms underlying damaged TM in glaucoma. In this study, to assess the possible role of abnormal Wnt signaling in steroid-induced glaucoma, we analyzed the effects of small-molecule Wnt signaling modulators on Dex-induced expression extracellular matrix proteins of primary human TM cells. While Dex-treated TM cells exhibited increased collagen and fibronectin expression, we found that Wnt signaling inhibitor 3235-0367 suppressed these Dex-induced effects. We therefore propose that Wnt signaling plays an important role in Dex-mediated impairment of TM cell functions. Moreover, the use of small-molecule Wnt signaling inhibitors to treat TM cells may provide an opportunity of restoring TM tissue in steroid-induced glaucoma.

Selection of suitable reference genes for quantitative real-time PCR in trabecular meshwork cells under oxidative stress.

Oxidative stress-induced dysfunction in trabecular meshwork (TM) cells is considered a major alteration that can lead to glaucoma. Hydrogen peroxide (H2O2) is the most widely used agent for inducing oxidation in TM cells in vitro. Quantitative real-time PCR (qPCR) is an important method for studying alterations in gene expression, and suitable (i.e. invariant) reference genes must be defined to normalize expression levels. In this study, eight common reference genes, i.e. PRS18, ACTB, B2M, GAPDH, PPIA, HPRT1, YWHAZ, and TBP, were evaluated for use in studies of H2O2-induced dysfunction in TM cells. Three established algorithms, geNorm, NormFinder, and BestKeeper, were used to analyze the reference genes. ACTB expression was least affected by H2O2 treatment in TM cells, and the combination of PPIA and HPRT1 was the most suitable gene pair for normalization. GAPDH and TBP were the most unstable genes and accordingly should be avoided in experiments with TM cells. These results provide a foundation for analyses of the mechanisms underlying glaucoma, and emphasize the importance of selecting suitable reference genes for qPCR studies.

Effect of Trypan Blue on the Survival of Cultured Trabecular Meshwork Cells

PURPOSE: To evaluate the effects of trypan blue (TB) on the survival of cultured human trabecular meshwork cells (HTMCs). METHODS: Primarily cultured HTMCs were exposed to 0.05, 0.10 or 0.50% TB for 1, 5 or 30 min. Cellular survival was assessed using the MTT assay and degree of apoptosis was analyzed with flow cytometry using annexin-V/propidium iodide double staining. RESULTS: Long-term exposure or high concentration of TB decreased the survival of HTMCs (p < 0.05). In flow cytometric analysis, exposure to 0.50% TB for 30 min increased the degree of apoptosis (p < 0.05). Commercial TB decreased cell survival after exposure for 5 min and increased the degree of apoptosis after exposure for 30 min (p < 0.05). CONCLUSIONS: TB may cause cellular damage of cultured HTMCs and apoptosis could be the underlying mechanism. In TB-assisted cataract surgery, TB should be used for the shortest time possible and removed completely.

Effects of dexamethasone and HA1077 on actin cytoskeleton and ß-catenin in cultured human trabecular meshwork cells.

AIM: To investigate the effects of dexamethasone (DEX) and 1-(5-isoquinolinesulfonyl)-homopiperazine (HA1077) on actin cytoskeleton and ß-catenin in cultured human trabecular meshwork (HTM) cells. METHODS: The HTM cells were separated from human eyeball and cultured in vitro. They were divided into control group, DEX (1×10-6 mol/L) group, HA1077 (3×10-5 mol/L) group, and DEX (1×10-6 mol/L) and HA1077 (3×10-5 mol/L) group. Actin cytoskeleton and ß-catenin in HTM cells of the four groups were examined by immunofluorescence and Western blot analyses. RESULTS: In DEX group, there were reorganization of actin cytoskeleton and formation of cross linked actin networks (CLANs), which were partially reversed in DEX and HA1077 group. DEX treatment also induced an increased expression of ß-catenin, which was obviously reduced in DEX and HA1077 group. Meanwhile, the cultured HTM cells in HA1077 group had lower expression of ß-catenin than that in the control group. CONCLUSION: Our results show that HA1077 can reverse the changes of actin organization and expression of ß-catenin induced by DEX in cultured HTM cells, suggesting that HA1077 may play an important role in increasing outflow and reducing intraocular pressure.

Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells.

The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 µM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM cells.

Ribonuclease 5 coordinates signals for the regulation of intraocular pressure and inhibits neural apoptosis as a novel multi-functional anti-glaucomatous strategy.

Glaucoma is a vision-threatening disorder characterized by progressive death of retinal ganglion cells (RGCs), although little is known about therapeutic milestones. Due to its complex and multifactorial pathogenesis, multipronged therapeutic approach is needed. Angiogenin (ANG), now called ribonuclease (RNase) 5, has been previously known as angiogenic factor and more recently its biologic activity is extended to promoting cell survival via its ribonucleolytic activity. Here, we revealed the defect of ANG in human glaucomatous trabecular meshwork (TM) cells and identified novel multiple functions of ANG as an anti-glaucomatous strategy. ANG was highly expressed in normal eyes and normal TM cells compared to glaucomatous TM cells. ANG induced intraocular pressure (IOP) lowering in rat models of both normal and elevated IOP, and as a possible mechanism, activated Akt-mediated signals for nitric oxide (NO) production, an important regulator of IOP in glaucomatous TM cell. Moreover, we demonstrated ANG-induced production of matrix metalloproteinase (MMP)-1 and -3 and rho-kinase inhibition for TM remodeling. For anti-glaucomatous defense optimization, ANG not only elicited immune-modulative pathways via indolamine 2,3-dioxygenase (IDO) activation in TM cells and suppression of Jurkat T cells, but also rescued neural stem cells (NSCs) from apoptosis induced by glaucomatous stress. These results demonstrate that novel multi-functional effects of ANG may have benefits against glaucoma in ocular tissues.

Effects of sulforaphane on thioredoxin expression in bovine trabecular meshwork cells / 中华实验眼科杂志

Background Recent studies have confirmed that sulforaphane (SFN) can activate multiple pathways,and promote the expression of the antioxidants in cells.Thioredoxin (Trx) plays an important role in maintaining the intracellular redox in the steady state.Objective This study was to investigate the effect and mechanism of SFN on Trx expression in bovine trabecular meshwork cells (BTMCs) cultured in vitro.Methods BTMCs were cultured in vitro and identified by morphological evaluation.The third generation of BTMCs were cultured in the medium with 0,10,20 and 30 μmol/L SFN for 30 minutes.Real-time PCR was applied to measure the expression of Trx mRNA in BTMCs.The BTMCs were randomly divided into normal control group,LY294002 group,U0126 group,SFN group,LY294002 +SFN group and U0126+SFN group.The expressions of Nrf2 protein and Trx protein in each group were measured by Western blot.Results The BTMCs was successfully cultured in vitro.The expressions of Trx mRNA were significantly different among the different concentrationss of SFN treatment (F=88.090,P＜0.01).The expressions of Trx protein and Nrf2 protein in the LY294002 +SFN group,U0126 +SFN group and SFN group were significantly higher than those in the normal control group (all at P ＜ 0.01).The expressions of Trx protein and Nrf2 protein in the LY294002+SFN group and U0126+SFN group were significantly higher than those in the SFN group (all at P＜0.01).Conelusions SFN can activate Nrf2 by phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathways,which can increase the expression level of Trx in BTMCs cultured in vitro.

Changes of MMP-3 and MMP-9 expressions on human trabecular cells following the exposure of laser associated with selective laser trabeculoplasty / 中华实验眼科杂志

Background Selective laser trabeculoplasty (SLT) can increase the outflow of aqueous humor and reduce the intraocular pressure of patients with open angle glaucoma,but its mechanism is unknown.To investigate the mechanism of SLT would improve the therapeutic effect of SLT.The aqueous outflow resistance in trabecular meshwork was affected by the extracellular matrix (ECM).Matrix metalloproteinase-3 (MMP-3) and MMP-9 were closely related to ECM degradation in trabecular meshwork.Objective This study was to observe the effects of SLT on the expressions MMP-3 and-9 in human trabecular ceils in vitro.Methods Immortallized human trabecular cells were cultured with pigment particles mixed suspension for 16 hours and incubated overnight.Then the cells were irradiated with Q switch frequency doubling 532 nm Nd:YAG laser to establish SLT-effective cells with the energy of 0.2 mJ,spot diameter of 400 μm and pulse duration of 3 ns.The expressions of MMP-3 mRNA and MMP-9 mRNA in the cells were detected by fluorescence quantitative real time PCR before and 1 hour and 4,8,12,28 hours after exposure of laser.The concentrations of MMP-3 and MMP-9 in the medium were assayed using ELISA 1 hour and 4,8,12,28 hours after exposure of laser and compared between the non-irradiation group and the irradiation group.Results The relative expressing levels of MMP-3 mRNA were 1.00,1.32±0.12,2.08±0.05,2.34±0.04,2.77± 0.05 and 2.49±0.27 in the non-irradiation group and irradiation for 1 hour and 4,8,12,28 hours after exposure of laser SLT,showing a significant difference among the groups (F =15.966,P=0.007),and relative expressing levels of MMP-3 mRNA were significantly higher in various time points after laser irradiation than those of the non-irradiation group (all at P＜0.05).The relative expressing levels of MMP-9 mRNA were 1.00,0.91 ±0.10,1.27 ± 0.07,1.46±0.07,1.69±0.09 and 0.87±0.09 in the non-irradiation group and irradiation for 1 hour and 4,8,12,28 hours after exposure of SLT,which was considerably different among the groups (F =30.526,P =0.005),and significant increased values were seen in the 4,8 and 12 hours after irradiation compared with the non-irradiation group (all at P＜0.05),with highest expression in the irradiation for 12-hour group.The concentrations of MMP-3 and MMP-9 proteins in medium were significantly increased in various time points after laser exposure in comparison with the control group (all at P＜0.05).Conclusions The expressions of MMP-3 and MMP-9 in human trabercuolar cells upregulate and the secretion ability of human trabecular cells to MMP-3 and MMP-9 proteins improves in early stage of SLT in vitro.However,these procedures gradually diminish with the lapse of time.

Epithelial mesenchymal transition-like phenomenon in trabecular meshwork cells.

The trabecular meshwork (TM) is a major component of the conventional outflow pathway and the excess extracellular matrix (ECM), and fibrosis in the TM causes increased outflow resistance. In this study, we first investigated the effects of several ECM components in the induction of an epithelial mesenchymal transition (EMT)-like phenomenon in TM cells. TM cells were isolated from cynomolgus monkeys (Macaca fascicularis). The cells were cultured in ECM-coated dishes and then subjected to both western blot analysis and immunocytochemistry to measure the levels of EMT-associated markers. Cell motility was assessed using wound healing and chemotaxis assays. We found that type I collagen, fibronectin and laminin induced the dissociation of cell-cell contact and elongation of actin stress fibers in the cultured monkey TM cells. In addition, following the same stimulation of the ECM, the expression of mesenchymal markers, such as fibronectin and α-smooth muscle actin, and the phosphorylation of Smad2 increased in the TM cells. Our results showed the significant acceleration of TM cellular motility following stimulation with type I collagen, fibronectin and laminin. These phenomena were inhibited by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. In addition, siRNA against paxillin was transfected to evaluate the association between paxillin and the EMT-like phenomenon. The knockdown of paxillin expression by transfection with siRNA blocked the EMT-like alteration of the cellular characteristics and chemotaxis toward transforming growth factor-ß2 in the cultured TM cells. Our results showed that the ECM-JNK-paxillin pathway induced an EMT-like phenomenon in TM cells, resulting in the abundant expression of fibronectin and activation of motility in TM cells. This EMT-like phenomenon could result in aberrant conditions in the aqueous outflow pathway in glaucomatous eyes.

Effects of platelet-derived growth factor-BB on the expression of matrix metalioproteinase-2 in trabecular meshwork cells in vitro / 中华实验眼科杂志

Background At present,a new drug,platelet-derived growth factor-BB (PDGF-BB),as a drug knife for the treatment of glaucoma is under study to expect it directly working on the trabecular meshwork without disrupting the normal physiological structure of anterior chamber angle,clearing the trabecular meshwork aqueous outflow channel so as to achieve the purpose of lowering intraocular pressure.Objective This study aimed to investigate the effect of PDGF-BB on matrix metalloproteinase-2 (MMP-2) expression in cultured bovine trabecular meshwork cells.Methods Trabcular tissue was obtained from fresh bovine eyes,and trabcular meshwork cells were cultured and passaged using explant method.Cultured cells were identified by morphological evaluation and neuronspecific enolase (NSE) staining.The third generation of cells were inoculated to 6-well plate,and different concentrations (0,5.0,12.5,25.0 μg/L) of PDGF-BB was added into the medium for 2 hours.Expression levels (A value) of MMP-2 mRNA (MMP-2 mRNA/β-actin) and protein in the cells were assayed by reverse transcription polymerase chain reaction (RT-PCR) and immunochemistry,respectively.Results Trabcular meshwork cells appeared 5-9 days after cultured.The third generation of cells presented with many process and showed the green influence in cytoplasm.MMP-2 mRNA/β-actin value (A) was 0.127 ± 0.026,0.147 ± 0.045,0.178 ± 0.053 and 0.222±0.062 in the O,5.0,12.5,25.0 μg/L PDGF-BB group,respectively,showing a significant difference among them (F =56.71,P＜0.05),and the MMP-2 mRNA/β3-actin value in the 5.0,12.5,25.0 μg/L PDGF-BB group was elevated in comparison with that of the 0 μg/L PDGF-BB group (all P＜0.05).The expression value (A value) of PDGF-BB protein in the cells was 446.12±13.81,1444.65±54.64,2086.18±73.18,3488.65±25.98 in the 0,5.0,12.5,25.0 μg/L PDGF-BB group,respectively,with a significant difference among the four groups (F=213.12,P＜0.01),and the expression value (A value) of PDGF-BB protein was gradually increased with the ascend of concentration of PDGF-BB(all P＜0.05).Conclusions PDGF-BB can promote the expression of MMP-2 in bovine trabcular meshwork cells in vitro in concentration-dependent manner.

Effect of a chloride channel inhibitor on proliferation and apoptosis of human trabecular meshwork cell / 中华实验眼科杂志

Background5-Nitro-2-(3-styrene-acrylic amine) benzoic acid ( NPPB),a chloride channel inhibitor,has a promoting effect on cell apoptosis in myocardial ischemia and reperfusion of domestic rabbit.The CIC chloride channel has been found in the ocular trabecular cells.However,the effect of NPPB on the shape and function of trabecular cells is unclear. Objective This study was performed to investigate the effect of NPPB on the proliferation,cell cycle progression and apoptosis of human trabecular meshwork cells.MethodsThe immortalized human trabcular cell strain was cultured,and logarithmic-phase cells were incubated in 96-well plates at a density of 1 ×106/ml.Different concentrations of NPPB (10,50,100 μ mol/L) were added to the medium,and the MTT assay was used to assess the growth and proliferation of the cells.Flow cytometry was used to evaluate the cell cycle.Then,100 mg/L 5-FU or 100 mg/L 5-FU + 100 μmol/L NPPB was used to induce cell apoptosis,which was assessed by Annexin V-PI.The membrane potential of mitochondria was examined using rhodamine 123 (△ψm).Results After 48 hours of treatment with NPPB,the abosorbency (A value) of the cells was gradually lowered with the increasing dose of NPPB,with significant differences among the 4 groups (F =7.230,P =0.006).Compared with the 10 μmol/L NPPB group,the A values were significantly declined in the 50 and 100 μmol/L NPPB groups (t =1.610,P =0.025 ;t =12.270,P =0.001 ).Forty-eight hours after exposure to NPPB,the percentage of cells in G0/G1 phase was increased and that in the S phase was decreased.The percentages of cells in different phases of cell cycle were significantly different in comparison with their control groups (without NPPB)( P＜0.05 ).Twenty-four and 48 hours after the treatment with 100 mg/L 5-FU,the apoptosis rates of the cells were raised in the 100 mg/L 5-FU group and 100 mg/L 5-FU + 100 μmol/L NPPB group compared to the without NPPB group (t24h =2.130,P =0.023;t48h =4.810,P=0.011 ) ;while that in the 100 mg/L 5-FU+100 μmol/L NPPB group was higher than the 100 mg/L 5-FU group ( t24 h =1.980,P =0.037 ; t48 h =1.290,P =0.028 ),and the mitochondrial membrane potential was lowered ( t24h =1.580,P =0.029 ; F48 h =6.200,P =0.015 ).Conclusions NPPB suppresses the proliferation of human trabecular cells and promotes the cells to enter S phase via the G1/S check point.In addition,ClC might be involved in an anti-apoptosis mechanism through the internal mitochondrial pathway.

Effect of soluble CD44 molecule on the expression of apoptosis regulatory protein bcl-2 associated death factor bad in human trabecular meshwork cell / 中华实验眼科杂志

BackgroundResearches demonstrated that the levels of soluble CD44 (sCD44)molecule in aqueous is significantly higher in primary open-angle glaucomous(POAG) eye than normal eye,but how the sCD44 would affect the expression of apoptosis protein in trabecular meshwork cells is below understanding.Objective The present study was to investigate the effect of sCD44 on the expression of regulatory proteins bcl-2 associated death factor bad in trabecular meshwork cells in the patients with POAG.MethodsHuman scleral tissue with trabecular meshwork were obtained from POAG patients during the surgery.The trabecular meshwork cells were primarily cultured by explant culture method and identified by immunochemistry.The third generation of cells were incubated with free-serum DMEM/F12 medium added differnt dosages of sCD44 (0,1,5,10,25,50 mg/L) for 48 hours.The expression of bad protein in cultured cells was detected using cell counting kit-8 (CCK-8) as the absorbance values at 490 nm(A,90 value),and the bad protein level in cultured cells was assayed by ELISA.ResultsThe cultured cells showed the positive response for laminin ( LM ),neuron specific enolase ( NSE ),fibronectin ( FN ) monoclonal antibodies.The CCK-8 assay showed that the A490 values of the trabecular meshwork cells in 0,1,5,10,25,50 μg/L of sCD44 groups were 0.2460±0.0019,0.1874±0.0015,0.1570±0.0016,0.1302±0.0019,0.1084±0.0018,0.0940±0.0020 respectively with a statistically significant difference among the 6 groups( F =14.922,P =0.000 ),and the A490 values in various dosages of sCD44 groups were significantly lower than the 0 μg/L sCD44 group (P=0.013,0.008,0.011,0.005,0.004).The ELISA assay showed that bad protein levels in 0,1,5,10,25,50 μg/L of sCD44 groups were ( 114.8461 ± 2.9560 ),( 137.8270 ± 2.4259 ),( 161.4194 ± 3.7381 ),( 170.9453 ± 3.2006 ),( 221.2252 ±4.3738 ),( 324.6167±4.4220) ng/L,showing a total difference among them ( F =16.610,P =0.000 ),and the bad protein levels in various dosages of sCD44 groups were significantly lower than the 0 μg/L sCD44 group( P =0.017,0.013,0.008,0.007,0.006).ConclusionssCD44 can contribute to the apoptosis of the trabecular meshwork cells in patients with POAG in certain dose range by regulating the apoptosis regulatory proteins bcl-2 associated death factor bad.

Effects of interleukin-1α on the expression of matrix metalloproteinases and tissue matrix metalloproteinase inhibitors in swine trabecular meshwork cells / 中华实验眼科杂志

BackgroundObstruction of aqueous humor out flow pathway or abnormality of the extracellular matrix( ECM ) of trabecular meshwork cells causes high intraocular pressure. The balance of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases(TIMPs) is critical for the metabolism of ECM. Interleukin1α(IL-1α) can influence outflow of aqueous humor by regulating MMPs level. Objective This study was to investigate the effect of interleukin-1α on the expression of MMP-2,MMP-3 and TIMP-I in cultured swine trabecular meshwork cells.Methods Swine sclera with trabecular meshwork tissue was isolated from 20 swine eyes and cultured with explant cultured method. Cultured cells were passaged and third generation cells were identified by fibronectin ( FN ) and laminin ( LN ) staining. After 24 hours of serum starvation, trabecular meshwork cells treated with IL-1α at the concentration of 10 mg/L were regarded as the IL group,and serum-free culture medium used to treat trabecular meshwork cells was regarded as the control group. The expression of MMP-2, MMP-3 and TIMP-1 proteins in trabecular meshwork cells were detected by immunohistochemistry,and the expression of MMP-2 mRNA, MMP-3 mRNA and TIMP-1 mRNA were detected by RT-PCR. The examination results were compared between the two groups. ResultsThe third generation of cells were positive for FN and LM. Compared with the control group, the expression levels of MMP-3 and TIMP-1 proteins(A value) in trabecular meshwork cells were significantly higher in the IL group than the control group(t=-7. 694,t =-5. 199,P＜0. 05) ,but no obvious difference was found in the expression of MMP-2 between the two groups( t=-2. 365, P＞0.05 ). The higher expression levels in MMP-3 mRNA and TIMP-1 mRNA (A value) in trabecular meshwork cells were seen in comparison with the control group (t =-3. 025,t=-1. 921 ,P＜0. 05). However,similar results were found in the expression of MMP-2 mRNA between the two groups(t =- 1. 173, P＞0.05 ). ConclusionsThe overexpression of MMP-3 and TIMP-1 proteins and their mRNA leads to the imbalance of MMP-3/TIMP-1 and promotes the decomposition of ECM in the trabecular meshwork, and therefore increases aqueous outflow.