Role of ID Proteins in BMP4 Inhibition of Profibrotic Effects of TGF-ß2 in Human TM Cells.

Purpose: Increased expression of TGF-ß2 in primary open-angle glaucoma (POAG) aqueous humor (AH) and trabecular meshwork (TM) causes deposition of extracellular matrix (ECM) in the TM and elevated IOP. Bone morphogenetic proteins (BMPs) regulate TGF-ß2-induced ECM production. The underlying mechanism for BMP4 inhibition of TGF-ß2-induced fibrosis remains undetermined. Bone morphogenic protein 4 induces inhibitor of DNA binding proteins (ID1, ID3), which suppress transcription factor activities to regulate gene expression. Our study will determine whether ID1and ID3 proteins are downstream targets of BMP4, which attenuates TGF-ß2 induction of ECM proteins in TM cells. Methods: Primary human TM cells were treated with BMP4, and ID1 and ID3 mRNA, and protein expression was determined by quantitative PCR (Q-PCR) and Western immunoblotting. Intracellular ID1 and ID3 protein localization was studied by immunocytochemistry. Transformed human TM cells (GTM3 cells) were transfected with ID1 or ID3 expression vectors to determine their potential inhibitory effects on TGF-ß2-induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression. Results: Basal expression of ID1-3 was detected in primary human TM cells. Bone morphogenic protein 4 significantly induced early expression of ID1 and ID3 mRNA (P < 0.05) and protein in primary TM cells, and a BMP receptor inhibitor blocked this induction. Overexpression of ID1 and ID3 significantly inhibited TGF-ß2-induced expression of fibronectin and PAI-1 in TM cells (P < 0.01). Conclusions: Bone morphogenic protein 4 induced ID1 and ID3 expression suppresses TGF-ß2 profibrotic activity in human TM cells. In the future, targeting specific regulators may control the TGF-ß2 profibrotic effects on the TM, leading to disease modifying IOP lowering therapies.

Identification and localization of lamina cribrosa cells in the human optic nerve head.

One of the central features of glaucoma is progressive cupping and excavation of the optic nerve head (ONH). Unmyelinated retinal ganglion cell (RGC) axons exit the eye through the ONH, which is supported by the lamina cribrosa (LC) consisting of plates of connective tissue with channels for bundles of RGC axons. The LC progressively remodels during glaucoma, but the cellular and molecular mechanisms responsible for this remodeling are poorly understood. Two major cell types have been isolated and cultured from the human ONH, which differ in their characteristics. Glial fibrillary acidic protein (GFAP) positive ONH astrocytes are the major cell type and are reactive in glaucoma. GFAP negative LC cells are the second major cell type isolated from the human ONH, and in contrast to ONH astrocytes, are α-smooth muscle actin (α-SMA) positive. Although a number of in vitro studies have been conducted with LC cells, to date there has been no direct evidence for these cells in situ in the human ONH. We used GFAP and α-SMA immunofluorescent staining of human eyes to clearly demonstrate the presence of not only ONH astrocytes within the human ONH, but also LC cells within the cribriform (e.g. laminar) plates/beams of the LC region. Both of these cell types likely play important roles in the homeostatic maintenance of the ONH and pathogenic changes that occur in primary open angle glaucoma (POAG).

In vitro and in vivo neuroprotective effects of cJun N-terminal kinase inhibitors on retinal ganglion cells.

BACKGROUND: The c-Jun N-terminal kinase (JNK) signaling pathway plays an important role in neuronal pathophysiology. Using JNK inhibitors, we examined involvement of the JNK pathway in cultured rat retinal ganglion cell (RGC) death and in mouse retinal ischemia/reperfusion (I/R) injury of the visual axis. The in vitro effects of JNK inhibitors were evaluated in cultured adult rat retinal cells enriched in RGCs. Retinal I/R was induced in C57BL/6J mice through elevation of intraocular pressure to 120 mmHg for 60 min followed by reperfusion. SP600125 was administered intraperitoneally once daily for 28 days. Phosphorylation of JNK and c-Jun in the retina was examined by immunoblotting and immunohistochemistry. The thickness of retinal layers and cell numbers in the ganglion cell layer (GCL) were examined using H&E stained retinal cross sections and spectral domain optical coherence tomography (SD-OCT). Retinal function was measured by scotopic flash electroretinography (ERG). Volumetric measurement of the superior colliculus (SC) as well as VGLUT2 and PSD95 expression were studied. RESULTS: JNK inhibitors SP600125 and TAT-JNK-III, dose-dependently and significantly (p < 0.05) protected against glutamate excitotoxicity and trophic factor withdrawal induced RGC death in culture. In the I/R model, phosphorylation of JNK (pJNK) in the retina was significantly (p < 0.05) increased after injury. I/R injury significantly (p < 0.05) decreased the thickness of retinal layers, including the whole retina, inner plexiform layer, and inner nuclear layer and cell numbers in the GCL. Administration of SP600125 for 28 days protected against all these degenerative morphological changes (p < 0.05). In addition, SP600125 significantly (p < 0.05) protected against I/R-induced reduction in scotopic ERG b-wave amplitude at 3, 7, 14, 21 and 28 days after injury. SP600125 also protected against the I/R-induced losses in volume and levels of synaptic markers in the SC. Moreover, the protective effects of SP600125 in the retina and SC were also detected even with only 7 days (Days 1-7 after I/R) of SP600125 treatment. CONCLUSIONS: Our results demonstrate the important role the JNK pathway plays in retinal degeneration in both in vitro and in vivo models and suggest that JNK inhibitors may be a useful therapeutic strategy for neuroprotection of RGCs in the retina.

C1q propagates microglial activation and neurodegeneration in the visual axis following retinal ischemia/reperfusion injury.

BACKGROUND: C1q represents the initiating protein of the classical complement cascade, however recent findings indicate pathway independent roles such as developmental pruning of retinal ganglion cell (RGC) axons. Furthermore, chronic neuroinflammation, including increased expression of C1q and activation of microglia and astrocytes, appears to be a common finding among many neurodegenerative disease models. Here we compare the effects of a retinal ischemia/reperfusion (I/R) injury on glial activation and neurodegeneration in wild type (WT) and C1qa-deficient mice in the retina and superior colliculus (SC). Retinal I/R was induced in mice through elevation of intraocular pressure to 120 mmHg for 60 min followed by reperfusion. Glial cell activation and population changes were assessed using immunofluorescence. Neuroprotection was determined using histological measurements of retinal layer thickness, RGC counts, and visual function by flash electroretinography (ERG). RESULTS: Retinal I/R injury significantly upregulated C1q expression in the retina as early as 72 h and within 7 days in the superficial SC, and was sustained as long as 28 days. Accompanying increased C1q expression was activation of microglia and astrocytes as well as a significantly increased glial population density observed in the retina and SC. Microglial activation and changes in density were completely ablated in C1qa-deficient mice, interestingly however there was no effect on astrocytes. Furthermore, loss of C1qa significantly rescued I/R-induced loss of RGCs and protected against retinal layer thinning in comparison to WT mice. ERG assessment revealed early preservation of b-wave amplitude deficits from retinal I/R injury due to C1qa-deficiency that was lost by day 28. CONCLUSIONS: Our results for the first time demonstrate the spatiotemporal changes in the neuroinflammatory response following retinal I/R injury at both local and distal sites of injury. In addition, we have shown a role for C1q as a primary mediator of microglial activation and pathological damage. This suggests developmental mechanisms of C1q may be re-engaged during injury response, modulation of which may be beneficial for neuroprotection.

Lysyl oxidases in the trabecular meshwork.

The mechanical properties of the extracellular matrix (ECM) play an important role in maintaining cellular function and overall tissue homeostasis. Emerging evidence suggests that biomechanical modifications of the ECM may be initiators and/or drivers of disease, exemplified by increased tissue stiffness. Specific ECM cross-linking enzymes (tissue transglutaminase, lysyl oxidase, and lysyl oxidase-like 1) are expressed in the trabecular meshwork and are regulated by transforming growth factor beta (TGF-ß) isoforms. As TGF-ß isoforms are elevated in the aqueous humor of glaucoma patients, trabecular meshwork stiffness mediated by ECM cross-linking may be responsible for increased aqueous humor outflow resistance and elevated intraocular pressure.

Monitoring retinal morphologic and functional changes in mice following optic nerve crush.

PURPOSE: We characterized the morphologic and functional changes in optic nerve crushed mice and evaluated electroretinogram (ERG) responses as tools to monitor retinal ganglion cell (RGC) dysfunction. METHODS: We performed optic nerve crush (ONC) unilaterally in adult BALB/cJ mice. The neuronal loss in the RGC layer (GCL) and superior colliculus (SC) was determined by Nissl staining. Retinal thickness was assessed by spectral-domain optical coherence tomography (SD-OCT) imaging. Retinal function was determined by pattern ERG and full-field flash ERG. Responses of pattern ERG, positive scotopic threshold response (pSTR), scotopic oscillatory potentials (OPs), and photopic negative response (PhNR) were analyzed. RESULTS: The ONC induced progressive neuronal loss in GCL and contralateral SC starting from 7 and 28 days following ONC, respectively. A linear correlation was observed between combined thickness of nerve fiber layer (NFL), GCL, and inner plexiform layer (IPL) imaged by SD-OCT and cell numbers in GCL. Only half of the normal BALB/cJ mice exhibited pattern ERG response, which was smaller and later compared to C57BL/6J mice. The ONC reduced pattern ERG and pSTR, but the reduction of pattern ERG was earlier than pSTR, preceding the anatomical cell loss in the GCL. The PhNR and scotopic OPs were not affected by ONC. CONCLUSIONS: The SD-OCT and ERG can be used to monitor noninvasively retinal morphologic and functional changes induced by ONC. Pattern ERG and pSTR are able to detect early RGC dysfunction, but pattern ERG exhibits higher sensitivity. Our results support the use of these tools in studies using the mouse ONC model.

Optic nerve crush induces spatial and temporal gene expression patterns in retina and optic nerve of BALB/cJ mice.

BACKGROUND: Central nervous system (CNS) trauma and neurodegenerative disorders trigger a cascade of cellular and molecular events resulting in neuronal apoptosis and regenerative failure. The pathogenic mechanisms and gene expression changes associated with these detrimental events can be effectively studied using a rodent optic nerve crush (ONC) model. The purpose of this study was to use a mouse ONC model to: (a) evaluate changes in retina and optic nerve (ON) gene expression, (b) identify neurodegenerative pathogenic pathways and (c) discover potential new therapeutic targets. RESULTS: Only 54% of total neurons survived in the ganglion cell layer (GCL) 28 days post crush. Using Bayesian Estimation of Temporal Regulation (BETR) gene expression analysis, we identified significantly altered expression of 1,723 and 2,110 genes in the retina and ON, respectively. Meta-analysis of altered gene expression (≥1.5, ≤-1.5, p < 0.05) using Partek and DAVID demonstrated 28 up and 20 down-regulated retinal gene clusters and 57 up and 41 down-regulated optic nerve clusters. Regulated gene clusters included regenerative change, synaptic plasticity, axonogenesis, neuron projection, and neuron differentiation. Expression of selected genes (Vsnl1, Syt1, Synpr and Nrn1) from retinal and ON neuronal clusters were quantitatively and qualitatively examined for their relation to axonal neurodegeneration by immunohistochemistry and qRT-PCR. CONCLUSION: A number of detrimental gene expression changes occur that contribute to trauma-induced neurodegeneration after injury to ON axons. Nrn1 (synaptic plasticity gene), Synpr and Syt1 (synaptic vesicle fusion genes), and Vsnl1 (neuron differentiation associated gene) were a few of the potentially unique genes identified that were down-regulated spatially and temporally in our rodent ONC model. Bioinformatic meta-analysis identified significant tissue-specific and time-dependent gene clusters associated with regenerative changes, synaptic plasticity, axonogenesis, neuron projection, and neuron differentiation. These ONC induced neuronal loss and regenerative failure associated clusters can be extrapolated to changes occurring in other forms of CNS trauma or in clinical neurodegenerative pathological settings. In conclusion, this study identified potential therapeutic targets to address two key mechanisms of CNS trauma and neurodegeneration: neuronal loss and regenerative failure.

Role of the alternatively spliced glucocorticoid receptor isoform GRß in steroid responsiveness and glaucoma.

Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious side effect of GC therapy in susceptible individuals. This OHT is due to increased aqueous humor (AH) outflow resistance in the trabecular meshwork (TM) caused by GC-mediated changes in TM structure and function. GCs may also play a role in the development of primary open-angle glaucoma (POAG). Elevated cortisol levels in the AH or enhanced GC sensitivity may be one of the reasons for elevated intraocular pressure in POAG patients. The GC OHT responder population is at greater risk of developing POAG compared with non-responders. We recently have gained insight into the molecular mechanisms responsible for this differential GC responsiveness, which is attributed to differences in GC receptor isoform expression in the TM. This article summarizes current knowledge on alternative GC receptor splicing to generate GC receptor alpha (GRα) and GRß and their roles in the regulation of GC responsiveness in normal and glaucoma TM.

The role of TGF-ß2 and bone morphogenetic proteins in the trabecular meshwork and glaucoma.

Primary open-angle glaucoma (POAG) is the second leading cause of blindness worldwide. Elevated intraocular pressure (IOP) is a primary risk factor associated with POAG. Increased aqueous humor (AH) outflow resistance through the trabecular meshwork (TM) results in elevated IOP in POAG patients. Resistance to AH outflow is associated with increased accumulation of extracellular matrix (ECM) proteins in the TM. In addition, levels of transforming growth factor-beta2 (TGF-ß2) are elevated in the AH and TM tissue of POAG patients. Elevated levels of TGF-ß2 in other tissues have been associated with fibrosis and increased tissue stiffness. However, locally produced effectors that maintain homeostatic relationships must also be present. Bone morphogenetic proteins (BMPs) serve this purpose in the TM as they inhibit TGF-ß2-induced ECM changes in TM cells. This review article first describes the TGF-ß superfamily of growth factors including BMPs and their canonical and noncanonical signaling pathways. The article then addresses the role of TGF-ß2 in the pathophysiology of POAG as related to the ECM and ECM crosslinking enzymes. This is followed by a discussion of potential homeostatic control mechanisms of TGF-ß2 signaling in the TM including the inhibitory role of BMP-4 and BMP-7. We then describe the relationship of TGF-ß2 and BMPs in TM fibrosis including the role of antagonists. Lastly, in future directions, we identify potential future studies that explore new and unique cellular interactions within the TM for potential therapeutic interventions.

Smad3 is necessary for transforming growth factor-beta2 induced ocular hypertension in mice.

TGFß2 induces extracellular matrix (ECM) remodeling and alters the cytoskeleton by both the canonical Smad and non-canonical signaling pathways. TGFß2 regulates the expression of ECM proteins in trabecular meshwork (TM) cells, increases intraocular pressure (IOP) in an ex vivo perfusion organ culture model, and induces ocular hypertension in rodent eyes. A necessary step in the canonical Smad signaling pathway is phosphorylation of receptor protein Smad3 by the TGF-ß receptor complex. The purpose of this study was to determine whether TGFß2 signals in vivo through the canonical Smad signaling pathway in the TM using Smad3 knockout (KO) mice. Ad5.hTGFß2(226/228) (2.5 × 10(7) pfu) was injected intravitreally into one eye of homozygous (WT), heterozygous (HET), and homozygous (KO) 129-Smad3(tm1Par)/J mice (n = 9-10 mice/group), with the uninjected contralateral eye serving as the control. IOP measurements were taken using a rebound tonometer. To test the effect of TGFß2 signaling on the ECM, fibronectin expression was determined by immunohistochemistry and qPCR analysis. Transduction of the TM with viral vector Ad5.hTGFß2(226/228) caused a statistically significant difference in IOP exposure between Smad3 genotypes: WT, 187.7 ± 23.9 mmHg*day (n = 9); HET, 95.6 ± 24.5 mmHg*day (n = 9); KO, 52.8 ± 25.2 mmHg*day (n = 10); (p < 0.05 WT versus HET, p < 0.01 WT versus KO). Immunohistochemistry and qPCR analysis showed that Ad5.hTGFß2(226/228) increased fibronectin expression in the TM of WT mice (2.23 ± 0.24 fold) compared to Smad3 KO mice (0.99 ± 0.19 fold), p < 0.05. These results demonstrate Smad3 is a necessary signaling protein for TGFß2-induced ocular hypertension and fibronectin deposition in the TM.

Cellular fibronectin expression in human trabecular meshwork and induction by transforming growth factor-ß2.

PURPOSE: Levels of TGF-ß2 are higher in POAG aqueous humor, causing deposition of extracellular matrix (ECM) proteins, including fibronectin (FN), in the glaucomatous human trabecular meshwork (HTM) that may be responsible for elevated IOP. The purpose of this study was to identify the expression of cellular FN (cFN) isoforms (EDA and EDB) in HTM cells and tissues, and to determine whether TGF-ß2 can induce cFN expression and fibril formation in cultured HTM cells. METHODS: Expression of cFN mRNA isoforms and induction by recombinant TGF-ß2 (5 ng/mL) were determined by quantitative RT-PCR. The TGF-ß2 induction of EDA isoform protein expression and FN fibril formation were analyzed using Western immunoblots and immunocytochemistry (ICC), respectively. Immunohistochemistry (IHC) analysis was used to examine total FN and EDA isoform expression in normal (NTM) and glaucomatous (GTM) trabecular meshwork (TM) tissues. RESULTS: Both cFN mRNA isoforms were expressed in cultured HTM cells and were induced by TGF-ß2 after 2, 4, and 7 days (P < 0.05). Similarly, EDA isoform protein and fibril formation were increased after 4 and 7 days of TGF-ß2 treatment. Finally, GTM tissues had significantly greater EDA isoform protein levels (1.7-fold, P < 0.05) compared to NTM tissues. CONCLUSIONS: This study demonstrated that cFN isoforms are expressed and induced in HTM cells by TGF-ß2. Also, increased EDA isoform protein levels were seen in GTM tissues. Our findings suggest that induction of cFN isoform expression in the TM ECM may be a novel pathologic mechanism involved in the TM changes associated with glaucoma.

Gremlin utilizes canonical and non-canonical TGFß signaling to induce lysyl oxidase (LOX) genes in human trabecular meshwork cells.

The TGFß/BMP signaling pathways are involved in glaucomatous damage to the trabecular meshwork (TM) leading to elevated intraocular pressure (IOP), which is a major risk factor for the development and progression of glaucoma. The BMP antagonist gremlin is elevated in glaucomatous TM cells and tissues and can directly elevate IOP. Gremlin utilizes the TGFß2/SMAD pathway to induce TM extracellular matrix (ECM) proteins. The purpose of this study is to determine whether expression of the ECM cross-linking lysyl oxidase (LOX) genes is regulated by gremlin in cultured human TM cells. Human TM cells were treated with recombinant gremlin, and expression of the LOX genes was examined by quantitative RT-PCR and western immunoblotting. TM cells were pretreated with TGFBR inhibitors (LY364947 or SB431542), an inhibitor of the SMAD signaling pathway (SIS3), or with JNK (SP600125) and p38 MAPK (SB203580) inhibitors to identify the signaling pathway(s) involved in gremlin induction of LOX protein expression. All five LOX genes (LOX and LOXL1-4) were induced by gremlin. Gremlin induction of LOX genes and protein expression was blocked by TGFBR inhibitors as well as by inhibitors of the SMAD3, JNK and p38 MAPK signaling pathways. We conclude that gremlin employs both canonical TGFß/SMAD and the non-canonical JNK and p38 MAPK signaling pathways to induce LOX genes and proteins in cultured human TM cells. Increased LOX levels may be at least partially responsible for gremlin-mediated IOP elevation and increased aqueous humor outflow resistance leading to glaucoma.

Progressive morphological changes and impaired retinal function associated with temporal regulation of gene expression after retinal ischemia/reperfusion injury in mice.

Retinal ischemia/reperfusion (I/R) injury is an important cause of visual impairment. However, questions remain on the overall I/R mechanisms responsible for progressive damage to the retina. In this study, we used a mouse model of I/R and characterized the pathogenesis by analyzing temporal changes of retinal morphology and function associated with changes in retinal gene expression. Transient ischemia was induced in one eye of C57BL/6 mice by raising intraocular pressure to 120 mmHg for 60 min followed by retinal reperfusion by restoring normal pressure. At various time points post I/R, retinal changes were monitored by histological assessment with H&E staining and by SD-OCT scanning. Retinal function was also measured by scotopic ERG. Temporal changes in retinal gene expression were analyzed using cDNA microarrays and real-time RT-PCR. In addition, retinal ganglion cells and gliosis were observed by immunohistochemistry. H&E staining and SD-OCT scanning showed an initial increase followed by a significant reduction of retinal thickness in I/R eyes accompanied with cell loss compared to contralateral control eyes. The greatest reduction in thickness was in the inner plexiform layer (IPL) and inner nuclear layer (INL). Retinal detachment was observed at days 3 and 7 post- I/R injury. Scotopic ERG a- and b-wave amplitudes and implicit times were significantly impaired in I/R eyes compared to contralateral control eyes. Microarray data showed temporal changes in gene expression involving various gene clusters such as molecular chaperones and inflammation. Furthermore, immunohistochemical staining confirmed Müller cell gliosis in the damaged retinas. The time-dependent changes in retinal morphology were significantly associated with functional impairment and altered retinal gene expression. We demonstrated that I/R-mediated morphological changes the retina closely associated with functional impairment as well as temporal changes in retinal gene expression. Our findings will provide further understanding of molecular pathogenesis associated with ischemic injury to the retina.

Transforming growth factor-ß2 induces expression of biologically active bone morphogenetic protein-1 in human trabecular meshwork cells.

PURPOSE: There are limited studies on the factors that regulate the processing of TGF-ß2 and extracellular matrix (ECM) proteins into their mature form. Bone morphogenic protein 1 (BMP1) is an enzyme responsible for the cleavage and maturation of growth factors and ECM proteins. The purpose of our study was to determine whether cultured human trabecular meshwork (TM) cells express BMP1, BMP1 expression is regulated by TGF-ß2, BMP1 is biologically active, and BMP1 regulates LOX activity. METHODS: Primary human TM cells were isolated and subjected to quantitative PCR (qPCR) and Western immunoblotting (WB) for BMP1. BMP1 immunolocalization was performed in TM tissues. qPCR was used to determine BMP1 mRNA expression and WB results were used to determine BMP1 protein expression. BMP1 activity was measured in TM cells treated with TGF-ß2 or with a combination of TGF-ß2/UK383367. Lysyl oxidase (LOX) enzyme activity was evaluated by WB in TM cells treated with BMP1 or with a combination of BMP1/ß-aminoprorionitrile (BAPN). RESULTS: Human TM cells expressed mRNA and protein for BMP1. Exogenous TGF-ß2 increased mRNA expression compared to their controls (P<0.05). An ELISA showed TGF-ß2-induced BMP1 secretion compared to their controls in all cell strains (P<0.05). Secreted BMP1 stimulated LOX enzymatic activity in TM cells. CONCLUSIONS: BMP1 is expressed in the human TM. TGF-ß2 induction of BMP1 may be responsible for increased processing of growth factors and ECM proteins into their mature forms, resulting in TM stiffness and resistance to ECM degradation.

The effect of postmortem time on the RNA quality of human ocular tissues.

PURPOSE: Profiling gene expression in human ocular tissues provides invaluable information for understanding ocular biology and investigating numerous ocular diseases. Accurate measurement of gene expression requires high-quality RNA, which often is a challenge with postmortem ocular tissues. METHODS: We examined the effect of various death to preservation (DP) times on the RNA quality of ten different ocular tissues. We used 16 eyes from eight different human donors. The eyes were preserved immediately in RNAlater or preserved after initial storage at 4 °C to create a range of DP times from 2 to 48 h. Ten ocular tissues were dissected from each eye. After total RNA was extracted from each dissected ocular tissue, the RNA integrity number (RIN) was determined using an Agilent Bioanalyzer. RESULTS: The RIN values from corneal and trabecular meshwork tissues were significantly (p<0.05) higher than those from the ciliary body at an earlier DP time (<6 h), but were not different among all tissues after 8 h. Interestingly, the RIN values from non-vascularized tissues were significantly (p=0.0002) higher than those from vascularized ocular tissues at early DP times (<6 h). The RIN value from the cornea was significantly (p<0.05) higher at short DP times compared to longer DP times. The RIN values from corneal tissues were significantly correlated to DP time according to regression analysis (p<0.05). CONCLUSIONS: In this study, we determined RNA quality from postmortem ocular tissues with various DP times. Our results emphasize the need for rapid preservation and processing of postmortem human donor eye tissues, especially for vascularized ocular tissues.

A magnetic bead-based method for mouse trabecular meshwork cell isolation.

PURPOSE: Mice have been used widely for glaucoma research. However, due to the small size of the mouse eye, it is difficult to dissect mouse trabecular meshwork (MTM) tissues and establish MTM cell strains. To circumvent this problem, we took advantage of the phagocytic property of trabecular meshwork (TM) cells, and developed a novel magnetic bead-based method that enables us to isolate pure MTM cells. METHODS: After anesthesia, up to 2 µL of fluorescent or magnetic microbeads were injected intracamerally into the mouse eyes. To study the distribution and localization of the beads, mice were sacrificed 1 to 7 days after injection, and eyes were enucleated for fluorescent or transmission electron microscopy (TEM) study, respectively. To isolate MTM cells, anterior segments injected with magnetic beads were dissected from 10 to 15 sterilized mouse eyes 7 days after injection. The tissues were digested with collagenase A and purified by using a magnetic field as well as repeated washing. RESULTS: TEM studies showed that the magnetic beads were located in the mouse TM, but not in corneal or scleral fibroblast cells. Cultured MTM cells were similar morphologically to human TM cells. MTM cells expressed TM markers, including collagen IV, laminin, and α-smooth muscle actin. Also, MTM cells treated with 100 nM dexamethasone showed increased formation of cross-linked actin networks and induction of myocilin expression. CONCLUSIONS: The magnetic bead-based method is efficient for isolating MTM cells with minimal microdissection techniques required. It will be a useful approach for isolating TM cells from small animals for glaucoma research.

Effects of thailanstatins on glucocorticoid response in trabecular meshwork and steroid-induced glaucoma.

PURPOSE: Elevated intraocular pressure (IOP) is a major risk factor in glaucoma. Various changes in the trabecular meshwork (TM) are responsible for elevated IOP. Glucocorticoids (GCs) increase IOP and mediate biochemical changes in the TM, similar to those associated with primary open-angle glaucoma (POAG). There are differences in steroid responsiveness among the population. Approximately 40% of individuals significantly elevate IOP (i.e., responders) upon GC administration, while others do not (i.e., nonresponders). The responders are at higher risk of developing POAG compared to the nonresponders. In addition, almost all POAG patients are steroid responders. GC responsiveness is regulated by the relative levels of the active GC receptor alpha (GRα) and the alternatively spliced dominant negative regulator isoform GRß. Glaucomatous TM cell strains have a lower GRß-GRα ratio compared to normal TM cells, making them more sensitive to GCs. Our purpose was to investigate the role of a special class of natural products called thailanstatins (TSTs) in GR alternative splicing and GC response in cultured human TM cells. METHODS: Quantitative RT-PCR and Western immunoblotting were used to study the effect of TSTs on GRß-GRα ratios in human TM cell strains. Effects of TSTs on dexamethasone (DEX) responsiveness were assessed by GRE-luciferase reporter activity assay and fibronectin (FN) induction in TM cells. RESULTS: TSTs increased the GRß-GRα ratio in TM cells. Increased GRß-GRα ratios were associated with decreased DEX-mediated FN induction and GRE-luciferase activity. CONCLUSIONS: TSTs modulate the GR splicing process to enhance GRß levels and thereby decrease the GC response in cultured human TM cells. These TSTs, or similar compounds, may potentially be new glaucoma therapeutic agents.

Characterization of a spontaneously immortalized bovine trabecular meshwork cell line.

Trabecular meshwork (TM) cells have widely been used as an in vitro model for glaucoma research. However, primary TM cells suffer the disadvantages of limited cell numbers and slow rates of proliferation. We discovered a spontaneously transformed bovine TM (BTM) cell line, BTM-28T. This cell line proliferated rapidly in low-glucose culture medium but also demonstrated contact inhibition in high-glucose culture medium. BTM-28T cells expressed key TM cell markers including α-smooth muscle actin (α-SMA), laminin and collagen IV (col IV). Also, 100 nM dexamethasone (DEX) enhanced the formation of cross-linked actin networks (CLANs) in confluent BTM-28T cell cultures. Transforming growth factor beta 2 (TGFß2) induced the expression of fibronectin (FN), plasminogen activator inhibitor-1 (PAI-1), and connective tissue growth factor (CTGF) in our cell cultures. This cell line will be helpful to better understand the aqueous humor outflow pathway as related to the pathophysiology of glaucoma.

Existence of the canonical Wnt signaling pathway in the human trabecular meshwork.

PURPOSE: We previously discovered elevated levels of secreted frizzled-related protein 1 (sFRP1), the Wnt signaling pathway inhibitor, in the glaucomatous trabecular meshwork (GTM), and found that key canonical Wnt signaling pathway genes are expressed in the trabecular meshwork (TM). The purpose of our study was to determine whether a functional canonical Wnt signaling pathway exists in the human TM (HTM). METHODS: Western immunoblotting and/or immunofluorescent microscopy were used to study ß-catenin translocation as well as the actin cytoskeleton in transformed and primary HTM cells. A TCF/LEF luciferase assay was used to study functional canonical Wnt signaling, which was confirmed further by WNT3a-induced expression of a pathway target gene, AXIN2, via quantitative PCR. Intravitreal injection of an Ad5 adenovirus expressing Dickkopf-related protein-1 (DKK1) was used to study the in vivo effect of canonical Wnt signaling on IOP in mice. RESULTS: WNT3a induced ß-catenin translocation in the HTM, which was blocked by co-treatment with sFRP1. Similarly, WNT3a enhanced luciferase levels in TCF/LEF luciferase assays, which also were blocked by sFRP1. Furthermore, AXIN2 expression was elevated significantly by WNT3a. However, neither WNT3a nor sFRP1 affected actin cytoskeleton organization, which theoretically could be regulated by noncanonical Wnt signaling in HTM cells. Exogenous DKK1, a specific inhibitor for the canonical Wnt signaling pathway, or sFRP1 elevated mouse IOP to equivalent levels. CONCLUSIONS: There is a canonical Wnt signaling pathway in the TM, and this canonical Wnt pathway, but not the noncanonical Wnt signaling pathway, regulates IOP.

The effects of transforming growth factor-ß2 on the expression of follistatin and activin A in normal and glaucomatous human trabecular meshwork cells and tissues.

PURPOSE: To compare follistatin (FST) and activin (Act) expression in normal and glaucomatous trabecular meshwork (TM) cells and tissues and determine if exogenous TGF-ß2 regulates the expression of FST and Act in TM cells. METHODS: Total RNA was isolated from TM cell strains, and mRNA expression for FST 317/344 isoforms and Act was determined via RT-PCR and quantitative PCR (qPCR). Western immunoblotting and immunocytochemistry determined FST and Act A protein levels in normal TM (NTM) and glaucomatous TM (GTM) cells. Cells were treated with recombinant human TGF-ß2 protein at 0 to 10 ng/mL for 0 to 72 hours. qPCR, Western immunoblotting, immunocytochemistry, and ELISA immunoassay were utilized to determine changes in FST and Act A mRNA and protein levels. In addition, NTM and GTM tissue samples were examined by immunohistochemistry for expression of FST, FST 315, FST 288, and Act A. RESULTS: Both FST mRNA and protein levels were significantly elevated in GTM cells. FST mRNA transcripts FST 317/344 were also significantly elevated in GTM cells. Immunohistochemistry showed FST levels were significantly elevated in GTM tissues. Exogenous TGF-ß2 significantly induced FST mRNA and protein expression. Immunohistochemistry demonstrated that Act A protein levels were significantly higher in NTM tissues compared to GTM tissues. CONCLUSIONS: FST is elevated in GTM cells and tissues. FST is known to be an inhibitor of bone morphogenetic proteins (BMPs), which, coupled with the ability of TGF-ß2 to upregulate FST levels, may indicate a possible role of FST in the pathogenesis of glaucoma. These results suggest that additional endogenous molecules in human TM may regulate TGF-ß2 signaling via inhibition of BMP family members.