Digital spatial profiling of segmental outflow regions in trabecular meshwork reveals a role for ADAM15.

In this study we used a spatial transcriptomics approach to identify genes specifically associated with either high or low outflow regions in the trabecular meshwork (TM) that could potentially affect aqueous humor outflow in vivo. High and low outflow regions were identified and isolated from organ cultured human anterior segments perfused with fluorescently-labeled 200 nm FluoSpheres. The NanoString GeoMx Digital Spatial Profiler (DSP) platform was then used to identified genes in the paraffin embedded tissue sections from within those regions. These transcriptome analyses revealed that 16 genes were statistically upregulated in high outflow regions and 57 genes were statistically downregulated in high outflow regions when compared to low outflow regions. Gene ontology enrichment analysis indicated that the top three biological categories of these differentially expressed genes were ECM/cell adhesion, signal transduction, and transcription. The ECM/cell adhesion genes that showed the largest differential expression (Log2FC ±1.5) were ADAM15, BGN, LDB3, and CRKL. ADAM15, which is a metalloproteinase that can bind integrins, was upregulated in high outflow regions, while the proteoglycan BGN and two genes associated with integrin signaling (LDB3, and CRKL) were downregulated. Immunolabeling studies supported the differential expression of ADAM15 and showed that it was specifically upregulated in high outflow regions along the inner wall of Schlemm's canal and in the juxtacanalicular (JCT) region of the TM. In addition to these genes, the studies showed that genes for decorin, a small leucine-rich proteoglycan, and the α8 integrin subunit were enriched in high outflow regions. These studies identify several novel genes that could be involved in segmental outflow, thus demonstrating that digital spatial profiling could be a useful approach for understanding segmental flow through the TM. Furthermore, this study suggests that changes in the expression of genes involved in regulating the activity and/or organization of the ECM and integrins in the TM are likely to be key players in segmental outflow.

NFATc1 Regulation of Dexamethasone-Induced TGFB2 Expression Is Cell Cycle Dependent in Trabecular Meshwork Cells.

Although elevated TGFß2 levels appear to be a causative factor in glaucoma pathogenesis, little is known about how TGFß2 expression is regulated in the trabecular meshwork (TM). Here, we investigated if activation of the cytokine regulator NFATc1 controlled transcription of TGFß2 in human TM cells by using dexamethasone (DEX) to induce NFATc1 activity. The study used both proliferating and cell cycle arrested quiescent cells. Cell cycle arrest was achieved by either cell-cell contact inhibition or serum starvation. ß-catenin staining and p21 and Ki-67 nuclear labeling were used to verify the formation of cell-cell contacts and activity of the cell cycle. NFATc1 inhibitors cyclosporine A (CsA) or 11R-VIVIT were used to determine the role of NFATc1. mRNA levels were determined by RT-qPCR. DEX increased TGFß2 mRNA expression by 3.5-fold in proliferating cells but not in quiescent cells or serum-starved cells, and both CsA and 11R-VIVIT inhibited this increase. In contrast, the expression of other DEX/NFATc1-induced mRNAs (myocilin and ß3 integrin) occurred regardless of the proliferative state of the cells. These studies show that NAFTc1 regulates TGFß2 transcription in TM cells and reveals a previously unknown connection between the TM cell cycle and modulation of gene expression by NFATc1 and/or DEX in TM cells.

Integrin Crosstalk and Its Effect on the Biological Functions of the Trabecular Meshwork/Schlemm's Canal.

Integrins are a family of heterodimeric receptors composed of an α- and ß-subunit that mediate cell-adhesion to a number of extracellular matrix (ECM) proteins in the Trabecular Meshwork/Schlemm's canal (TM/SC) of the eye. Upon binding an ECM ligand, integrins transmit signals that activate a number of signaling pathways responsible for regulating actin-mediated processes (i.e phagocytosis, cell contractility, and fibronectin fibrillogenesis) that play an important role in regulating intraocular pressure (IOP) and may be involved in glaucoma. An important function of integrin-mediated signaling events is that the activity of one integrin can affect the activity of other integrins in the same cell. This creates a crosstalk that allows TM/SC cells to respond to changes in the ECM presumably induced by the mechanical forces on the TM/SC, aging and disease. In this review, we discuss how integrin crosstalk influences the function of the human TM/SC pathway. In particular, we will discuss how different crosstalk pathways mediated by either the αvß3 or α4ß1 integrins can play opposing roles in the TM when active and therefore act as on/off switches to modulate the cytoskeleton-mediated processes that regulate the outflow of aqueous humor through the TM/SC.

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Overexpression and Activation of αvß3 Integrin Differentially Affects TGFß2 Signaling in Human Trabecular Meshwork Cells.

Studies from our laboratory have suggested that activation of αvß3 integrin-mediated signaling could contribute to the fibrotic-like changes observed in primary open angle glaucoma (POAG) and glucocorticoid-induced glaucoma. To determine how αvß3 integrin signaling could be involved in this process, RNA-Seq analysis was used to analyze the transcriptomes of immortalized trabecular meshwork (TM) cell lines overexpressing either a control vector or a wild type (WT) or a constitutively active (CA) αvß3 integrin. Compared to control cells, hierarchical clustering, PANTHER pathway and protein-protein interaction (PPI) analysis of cells overexpressing WT-αvß3 integrin or CA-αvß3 integrin resulted in a significant differential expression of genes encoding for transcription factors, adhesion and cytoskeleton proteins, extracellular matrix (ECM) proteins, cytokines and GTPases. Cells overexpressing a CA-αvß3 integrin also demonstrated an enrichment for genes encoding proteins found in TGFß2, Wnt and cadherin signaling pathways all of which have been implicated in POAG pathogenesis. These changes were not observed in cells overexpressing WT-αvß3 integrin. Our results suggest that activation of αvß3 integrin signaling in TM cells could have significant impacts on TM function and POAG pathogenesis.

Disruption of fibronectin fibrillogenesis affects intraocular pressure (IOP) in BALB/cJ mice.

Increased deposition of fibronectin fibrils containing EDA+fibronectin by TGFß2 is thought to be involved in the reduction of aqueous humor outflow across the trabecular meshwork (TM) of the eye and the elevation in intraocular pressure (IOP) observed in primary open angle glaucoma (POAG). Using a fibronectin-binding peptide called FUD that can disrupt fibronectin fibrillogenesis, we examined if disrupting fibronectin fibrillogenesis would affect IOP in the TGFß2 BALB/cJ mouse model of ocular hypertension. BALB/cJ mice that had been intravitreally injected with an adenovirus (Ad5) expressing a bioactive TGFß2226/228 showed a significant increase in IOP after 2 weeks. When 1µM FUD was injected intracamerally into mice 2 weeks post Ad5-TGFß2 injection, FUD significantly reduced IOP after 2 days. Neither mutated FUD (mFUD) nor PBS had any effect on IOP. Four days after FUD was injected, IOP returned to pre-FUD injection levels. In the absence of TGFß2, intracameral injection of FUD had no effect on IOP. Western blotting of mouse anterior segments expressing TGFß2 showed that FUD decreased fibronectin levels 2 days after intracameral injection (p<0.05) but not 7 days compared to eyes injected with PBS. mFUD injection had no significant effect on fibronectin levels at any time point. Immunofluorescence microscopy studies in human TM (HTM) cells showed that treatment with 2ng/ml TGFß2 increased the amount of EDA+ and EDB+ fibronectin incorporated into fibrils and 2µM FUD decreased both EDA+ and EDB+ fibronectin in fibrils. An on-cell western assay validated this and showed that FUD caused a 67% reduction in deoxycholate insoluble fibronectin fibrils in the presence of TGFß2. FUD also caused a 43% reduction in fibronectin fibrillogenesis in the absence of TGFß2 while mFUD had no effect. These studies suggest that targeting the assembly of fibronectin fibrillogenesis may represent a way to control IOP.

Role of Fibronectin in Primary Open Angle Glaucoma.

Primary open angle glaucoma (POAG) is the most common form of glaucoma and the 2nd most common cause of irreversible vision loss in the United States. Nearly 67 million people have the disease worldwide including >3 million in the United States. A major risk factor for POAG is an elevation in intraocular pressure (IOP). The increase in IOP is believed to be caused by an increase in the deposition of extracellular matrix proteins, in particular fibronectin, in a region of the eye known as the trabecular meshwork (TM). How fibronectin contributes to the increase in IOP is not well understood. The increased density of fibronectin fibrils is thought to increase IOP by altering the compliance of the trabecular meshwork. Recent studies, however, also suggest that the composition and organization of fibronectin fibrils would affect IOP by changing the cell-matrix signaling events that control the functional properties of the cells in the trabecular meshwork. In this article, we will discuss how changes in the properties of fibronectin and fibronectin fibrils could contribute to the regulation of IOP.

Activation of αvß3 Integrin Alters Fibronectin Fibril Formation in Human Trabecular Meshwork Cells in a ROCK-Independent Manner.

Purpose: Fibronectin fibrillogenesis is an integrin-mediated process that may contribute to the pathogenesis of primary open-angle glaucoma (POAG). Here, we examined the effects of αvß3 integrins on fibrillogenesis in immortalized TM-1 cells and human trabecular meshwork (HTM) cells. Methods: TM-1 cells overexpressing wild-type ß3 (WTß3) or constitutively active ß3 (CAß3) integrin subunits were generated. Control cells were transduced with an empty vector (EV). Deoxycholic acid (DOC) extraction of monolayers, immunofluorescence microscopy, and On-cell western analyses were used to determine levels of fibronectin fibrillogenesis and fibronectin fibril composition (EDA+ and EDB+ fibronectins) and conformation. αvß3 and α5ß1 Integrin levels were determined using fluorescence-activated cell sorting (FACS). Cilengitide and an adenovirus vector expressing WTß3 or CAß3 integrin subunits were used to examine the role of αvß3 integrin in HTM cells. The role of the canonical α5ß1 integrin-mediated pathway in fibrillogenesis was determined using the fibronectin-binding peptide FUD, the ß1 integrin function-blocking antibody 13, and the Rho kinase (ROCK) inhibitor Y27632. Results: Activation of αvß3 integrin enhanced the assembly of fibronectin into DOC-insoluble fibrils in both TM-1 and HTM cells. The formation of fibronectin fibrils was dependent on α5ß1 integrin and could be inhibited by FUD. However, fibrillogenesis was unaffected by Y27632. Fibrils assembled by CAß3 cells also contained high levels of EDA+ and EDB+ fibronectin and fibronectin that was stretched. Conclusions: αvß3 Integrin signaling altered the deposition and structure of fibronectin fibrils using a ß1 integrin/ROCK-independent mechanism. Thus, αvß3 integrins could play a significant role in altering the function of fibronectin matrices in POAG.

Genomic/proteomic analyses of dexamethasone-treated human trabecular meshwork cells reveal a role for GULP1 and ABR in phagocytosis.

Purpose: The purpose of this study is to examine the expression profile of genes related to integrin-mediated phagocytosis that are altered by dexamethasone (DEX) and/or αvß3 integrin signaling to gain a better understanding of the molecular basis of phagocytosis and the pathophysiology of glucocorticoid-induced ocular hypertension. Methods: RNA and cell lysates were obtained from human trabecular meshwork (HTM) cells incubated with and without DEX for 4-5 d. The relative level of gene expression was evaluated using the Affymetrix Gene Chip® human gene microarray and quantitative PCR (qPCR). Changes in protein expression were validated using western blots or FACS analyses. The involvement of proteins in phagocytosis was determined using siRNA to knock down the expression of these proteins in an immortalized TM-1 cell line. Changes in the phagocytic activity were measured using pHrodo™-labeled S. aureus bioparticles followed by immunofluorescence microscopy. The effect of αvß3 integrin expression and activity on GULP1 mRNA levels was measured using qPCR in TM-1 cells overexpressing wild type or constitutively active αvß3 integrin. Results: Gene microarrays revealed statistically significant differences (>2 fold) in the expression of seven genes known to be involved in phagocytosis. Three genes (CD36, ABR, and GULP1) were downregulated, while four genes (ITGB3, CHN1, PIK3R1, and MFGE8) were upregulated. The genes were either associated with modulating RAC1 activity (ABR and CHN1) or integrin signaling (CD36, GULP1, ITGB3, PIK3R1, and MFGE8). Another gene, SIRPA, was also downregulated (1.6 fold) but only in one cell strain. qPCR and western blot analyses verified that DEX caused a decrease in SIRPA and GULP1 mRNA and their protein levels, while levels of CHN1 mRNA and its protein were upregulated by DEX. qPCR showed that although ABR mRNA was downregulated compared to non-treated controls after 5 d of treatment with DEX, no change at the protein level was detected. qPCR analysis also revealed that DEX caused an increase in MFGE8 mRNA levels. The levels of CD36 mRNA and protein varied between cell strains treated with DEX and were not statistically different compared to controls. The knockdown of GULP1 and ABR using siRNAs decreased phagocytosis by 40%. Interestingly, GULP1 mRNA levels were also decreased by 60% when αvß3 integrin was overexpressed in TM-1 cells. Conclusion: The DEX-induced inhibition of phagocytosis may be caused by the downregulation of ABR and GULP1 disrupting the αvß5 integrin/RAC1-mediated engulfment pathway. The downregulation of GULP1 by αvß3 integrin further suggests that this integrin may be a negative regulator of phagocytosis by transcriptionally downregulating proteins needed for phagocytosis. In summary, these results represent new insights into the effects of glucocorticoids and integrin signaling on the phagocytic process in the TM.

Effect of αvß3 Integrin Expression and Activity on Intraocular Pressure.

Purpose: To determine the effects of αvß3 integrin expression and activation on intraocular pressure (IOP). Methods: Cre+/-ß3flox/flox mice were treated with topical tamoxifen eye drops for 5 days to activate Cre and excise the ß3 integrin gene from the anterior segment. IOP was measured weekly for 11 weeks using rebound tonometry. Mice were then killed and changes in expression of the ß3 integrin subunit in Cre+/- ß3flox/flox mice were determined using Western blotting analysis and immunofluorescence microscopy. To determine the effect of αvß3 integrin activation on outflow facility, porcine organ culture anterior segments (POCAS) were perfused with the αvß3 integrin-activating antibody AP5 or an isotype IgG control for 21 hours. The effect of αvß3 integrin activation on IOP was measured over 7 days in C57BL/6J mice intracamerally infused with AP5, AP3, IgG, or PBS. Results: Deletion of the ß3 integrin subunit using the tamoxifen-inducible Cre-loxP system resulted in a decrease in expression of the ß3 integrin subunit in the trabecular meshwork and ciliary muscle. Morphologically no gross changes in the anterior segment were detected. Deletion of the ß3 integrin subunit resulted in a significantly (P < 0.05) lower IOP in mice within 2 weeks following the tamoxifen treatment and persisted for 11 weeks. Activating the αvß3 integrin with the AP5 antibody resulted in a significant (P < 0.05) increase in IOP in C57BL/6J mice and a decrease in outflow facility in 42% of the POCAS. Conclusions: These studies demonstrate a role for αvß3 integrin signaling in the regulation of IOP.

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Absence of a secondary glucocorticoid response in C57BL/6J mice treated with topical dexamethasone.

Glucocorticoids such as dexamethasone can cause an increase in intraocular pressure (IOP) in some of the population, but not all. In this paper we used a mouse model of glucocorticoid induced ocular hypertension to examine the changes in the anterior segment of the eye in mice that failed to respond to glucocorticoid treatment with a sustained increase in IOP. C57BL/6J mice were treated with either 0.1% dexamethasone sodium phosphate ophthalmic solution or sterile PBS 3 times daily for up to 5 weeks. IOP was measured weekly at approximately the same time of the day. After 3-5 weeks of treatment, eyes were enucleated and evaluated for changes associated with steroid induced glaucoma. These studies showed that IOP was significantly elevated in dexamethasone (DEX) treated mice compared to PBS treated mice after 3 weeks of treatment, but IOP in DEX treated mice returned to baseline levels after 5 weeks of treatment. All the mice demonstrated a response to the glucocorticoid treatments and showed an elevation in FKBP5 expression after both 3 and 5 weeks of DEX treatment (primary glucocorticoid response protein) and a weight loss. Western blot analysis of anterior segments from treated mice, however, did not show an increase in secondary glucocorticoid response proteins such as ß3 integrin or myocilin. Fibronectin levels were also not statistically different. The data suggest that in mice, which do not exhibit a prolonged increase in IOP in response to the DEX treatment, there is a compensatory mechanism that can prevent or turn off the secondary glucocorticoid response.

The role of integrins in glaucoma.

Integrins are a family of heterodimeric transmembrane receptors that mediate adhesion to the extracellular matrix (ECM). In addition to their role as adhesion receptors, integrins can act as ''bidirectional signal transducers'' that coordinate a large number of cellular activities in response to the extracellular environment and intracellular signaling events. This bidirectional signaling helps maintain tissue homeostasis. Dysregulated bidirectional signaling, however, could trigger the propagation of feedback loops that can lead to the establishment of a disease state such as glaucoma. Here we discuss the role of integrins and bidirectional signaling as they relate to the glaucomatous phenotype with special emphasis on the αvß3 integrin. We present evidence that this particular integrin may have a significant impact on the pathogenesis of glaucoma.

NFATc1 activity regulates the expression of myocilin induced by dexamethasone.

Mutations in the myocilin gene (MYOC) account for 10% of juvenile open-angle glaucoma cases and 3-4% of adult onset primary open-angle glaucoma cases. It is a secreted glycoprotein found in many ocular and non-ocular tissues and has been linked to elevated intraocular pressure. In human trabecular meshwork (HTM) cells, MYOC expression can be induced by the glucocorticoid dexamethasone (DEX). In this study we examined the role of the calcineurin/NFATc1 (Nuclear Factor of Activated T-cells) pathway in the DEX induction of MYOC in HTM cells. In post-confluent HTM cells treated with either 500 nM DEX or 0.1% ethanol (EtOH; vehicle control) for 0-6 days both protein and mRNA levels of MYOC were increased while DEX was present. The protein and mRNA levels remained elevated for an additional 12 days after the removal of DEX. Only 1 day of DEX treatment was sufficient to trigger a sustained increase in MYOC mRNA that lasted for 4 days after the removal of DEX. Similar to other studies, myocilin protein expression was not seen until the second day of DEX treatment while mRNA increased within one day of DEX indicating that this is a secondary glucocorticoid response. To determine if MYOC gene expression was regulated by calcineurin/NFATc1, HTM cells were pre-treated for 1 h with the calcineurin inhibitors cyclosporin A or INCA-6 prior to the addition of DEX or EtOH for 2 days. NFATc1 siRNA was used to determine if NFATc1 was required for MYOC mRNA expression. Cells were also treated with the ionophone ionomycin to determine if increased cytosolic calcium affected MYOC expression. These studies showed that the DEX induced increase in MYOC mRNA could be inhibited with either cyclosporin A or INCA-6 or by transfection with NFATc1 siRNA and that ionomycin was unable to increase MYOC mRNA. Immunofluorescence microscopy was also performed to determine if DEX caused the nuclear translocation of NFATc1. Immunostaining showed that NFATc1 relocated to the nucleus within 15 min of DEX treatment and remained there for up to 2 h. The data suggest that the DEX-induced increase in MYOC expression activates a calcineurin and NFATc1 pathway in a calcium independent mechanism.

The role of integrins in the trabecular meshwork.

Integrins are a family of heterodimeric transmembrane receptors that mediate adhesion to the extracellular matrix (ECM). However, integrins are not just adhesion receptors. They can act as "bidirectional signal transducers" that coordinate a large number of cellular activities in response to the extracellular environment and intracellular signaling events. Among the activities regulated by integrins are cell adhesion, assembly of the ECM, growth factor signaling, apoptosis, organization of the cytoskeleton, and cytoskeleton-mediated processes such as contraction, endocytosis, and phagocytosis. Integrins regulate these activities through a complex network of intracellular signaling kinases and adaptor proteins that associate with the transmembrane and cytoplasmic domains of the integrin subunits. In this review, we will discuss how some of the known integrin-mediated activities can control the function of the trabecular meshwork. We will also discuss how integrin activity is a tightly regulated process that involves conformation changes within the heterodimer which are mediated by specific integrin-binding proteins.

Dexamethasone increases αvß3 integrin expression and affinity through a calcineurin/NFAT pathway.

The purpose of this study was to determine how dexamethasone (DEX) regulates the expression and activity of αvß3 integrin. FACS analysis showed that DEX treatment induced expression of an activated αvß3 integrin. Its expression remained high as long as DEX was present and continued following DEX removal. FACS analysis showed that the upregulation of αvß3 integrin was the result of an increase in the expression of the ß3 integrin subunit. By real time qPCR, DEX treatment induced a 6.2-fold increase (p<0.04) in ß3 integrin mRNA by day 2 compared to control and remained elevated for 6days of treatment and then an additional 10days once the DEX was removed. The increase in ß3 integrin mRNA levels required only 1day of DEX treatment to increase levels for 4days in the absence of DEX. In contrast, DEX did not alter ß1 integrin mRNA or protein levels. The DEX-induced upregulation of ß3 integrin mRNA was partly due to an increase in its half-life to 60.7h from 22.5h in control cultures (p<0.05) and could be inhibited by RU486 and cycloheximide, suggesting that DEX-induced de novo protein synthesis of an activation factor was needed. The calcineurin inhibitors cyclosporin A (CsA) and FK506 inhibited the DEX induced increase in ß3 integrin mRNA. In summary, the DEX-induced increase in ß3 integrin is a secondary glucocorticoid response that results in prolonged expression of αvß3 integrin and the upregulation of the ß3 integrin subunit through the calcineurin/NFAT pathway.

Comparative genomic and proteomic analysis of cytoskeletal changes in dexamethasone-treated trabecular meshwork cells.

Changes in the actin cytoskeleton, especially the formation of cross-linked actin networks (CLANs) are thought to contribute to the increased intraocular pressure observed in primary open-angle and steroid-induced glaucoma. To better understand the effects of glucocorticoids, we employed a shotgun method to analyze global changes in the cytoskeleton and integrin signaling pathways following dexamethasone (DEX) treatment of human trabecular meshwork (HTM) cells. RNA and cell lysates were obtained from HTM cells incubated with or without DEX. Changes in protein expression were determined by mass spectrometry (MS) following differential centrifugation of cell lysates to enrich for low-abundance cytoskeletal and signaling proteins, proteolytic digestion, and a titanium dioxide column to enrich for phosphopeptides. Results were validated by Western blots. Changes in RNA levels were determined with gene arrays and RT-PCR. Overall, MS identified 318 cytoskeleton associated proteins. Five of these proteins (PDLIM1, FGFR1OP, leiomodin-1, ZO-2 and LRP16A) were only detected in DEX-treated cells by MS. However, only PDLIM1 showed a statistically significant increase at the RNA level. Other proteins with differences at both the RNA and protein levels included ß3 integrin, caveolin-1, Borg2, raftlin1, PI-3 kinase regulatory subunit α, transgelin, and filamin B. By immunofluorescence microscopy filamin B and PDLIM1 showed enhanced expression in human trabecular meshwork cells, but only PDLIM1 demonstrated significant localization within CLANs. Finally, MS showed that some of the cytoskeleton proteins (Borg2, leiomodin-1, LRP16A, raftlin1 and CKAP4) contained phosphorylated residues. This study suggests that DEX affects the expression of cytoskeleton proteins at the transcriptional and translational level and shows that a combined genomic and proteomic approach can be used for rapid analysis of proteins in the TM. It also shows that DEX altered the expression of components (PDLIM1 and ß3 integrins) involved in CLAN formation and provides new findings into the effects of glucocorticoids on the cytoskeleton.

Integrin-linked kinase regulates integrin signaling in human trabecular meshwork cells.

PURPOSE: To determine whether integrin-linked kinase (ILK) controls the organization of the actin cytoskeleton in the trabecular meshwork (TM) by regulating integrin co-signaling. METHODS: The cell binding domain and the Heparin II (Hep II) domain of fibronectin were used to activate α5ß1 and α4ß1 integrin signaling, respectively, in differentiated human TM (HTM) cells. The role of ILK was determined using either ILK small interfering RNA (siRNA) to knockout ILK expression or the ILK inhibitors, KP392 and QLT0267. The knockdown of ILK expression was verified by Western blot analysis. The presence of actin stress fibers and focal adhesions was determined by labeling cultures with phalloidin and anti-talin or ILK antibodies, respectively. RESULTS: Cell spreading in differentiated HTM cells required ILK, since ILK siRNA and the ILK inhibitors significantly reduced cell spreading, actin polymerization, and the localization of talin and ILK in focal adhesions (FAs). Both cell spreading and the localization of talin and ILK to FAs in differentiated HTM cells could be rescued by inducing α4ß1 integrin signaling with a recombinant Hep II domain of fibronectin, even though α4ß1 integrins were not found in FAs. In the absence of ILK inhibition, the Hep II domain had minimal effect on α5ß1 integrin-mediated spreading. CONCLUSIONS: This study suggests that cooperative α5ß1/α4ß1 integrin signaling may be regulated by ILK trans-dominantly and that alterations in ILK activity may affect actin cytoskeleton organization and contractility in the TM.

COCH transgene expression in cultured human trabecular meshwork cells and its effect on outflow facility in monkey organ cultured anterior segments.

Purpose. To determine the effects of COCH transgene expression on cultured human trabecular meshwork (HTM) cell morphology and on outflow facility (OF) in monkey organ cultured anterior segments (MOCAS). Methods. An adenoviral (Ad) vector expressing both cochlin (COCH) and green fluorescent protein (GFP) (AdCOCHGFP) or GFP alone (AdGFP) was used to transduce cultured HTM cells (multiplicity of transduction, 2.8 and 28). COCH transgene expression in transduced HTM cells and the culture medium was verified by Western blot analysis and immunofluorescence detection 5 days after transduction. MOCAS were used to test the effect of Ad vectors (2.8 x 10(10) viral particles per segment) on OF. The morphology of transduced MOCAS was evaluated by light microscopy. Results. Western blot analysis showed a viral vector dose-dependent expression of cochlin in transduced cells and the culture medium. There was no notable morphologic change in transduced cells. In MOCAS, cochlin expression was detectable in the medium by 3 days after transduction. A 35% decrease in OF in AdCOCHGFP-transduced MOCAS was detected after 3 days, decreasing by 76% after 12 days when compared to control segments injected with AdGFP. Anterior segment pressure (ASP) more than doubled (P < 0.05) in segments injected with AdCOCHGFP at 12 days after transduction. Light microscopy revealed normal angle structures in transduced segments. Conclusions. Ad vector delivery of the COCH transgene resulted in cochlin expression in HTM cells and MOCAS. Cochlin expression was effective in decreasing OF and increasing ASP in MOCAS, suggesting possible involvement of cochlin in IOP elevation in vivo. COCH gene delivery has potential for use in developing a glaucoma model.

Functional properties of fibronectin in the trabecular meshwork.

Fibronectin plays a number of important roles in the extracellular matrix (ECM) including providing structural support and signaling cues for cell survival, migration, differentiation, gene expression, growth factor signaling, and cell contractility. In this review, we examine recent findings about the biological and structural properties of fibronectin and discuss how these properties could contribute to the regulation of aqueous humor (AH) outflow in the trabecular meshwork (TM).