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.

The Effects of Mechanical Stretch on Integrins and Filopodial-Associated Proteins in Normal and Glaucomatous Trabecular Meshwork Cells.

The trabecular meshwork (TM) is the tissue responsible for regulating aqueous humor fluid egress from the anterior eye. If drainage is impaired, intraocular pressure (IOP) becomes elevated, which is a primary risk factor for primary open angle glaucoma. TM cells sense elevated IOP via changes in their biomechanical environment. Filopodia cellular protrusions and integrin transmembrane proteins may play roles in detecting IOP elevation, yet this has not been studied in detail in the TM. Here, we investigate integrins and filopodial proteins, such as myosin-X (Myo10), in response to mechanical stretch, an in vitro technique that produces mechanical alterations mimicking elevated IOP. Pull-down assays showed Myo10 binding to α5 but not the ß1 subunit, αvß3, and αvß5 integrins. Several of these integrins colocalized in nascent adhesions in the filopodial tip and shaft. Using conformation-specific antibodies, we found that ß1 integrin, but not α5 or αvß3 integrins, were activated following 1-h mechanical stretch. Cadherin -11 (CDH11), a cell adhesion molecule, did not bind to Myo10, but was associated with filopodia. Interestingly, CDH11 was downregulated on the TM cell surface following 1-h mechanical stretch. In glaucoma cells, CDH11 protein levels were increased. Finally, mechanical stretch caused a small, yet significant increase in Myo10 protein levels in glaucoma cells, but did not affect cellular communication of fluorescent vesicles via filopodia-like tunneling nanotubes. Together, these data suggest that TM cell adhesion proteins, ß1 integrin and CDH11, have relatively rapid responses to mechanical stretch, which suggests a central role in sensing changes in IOP elevation in situ.

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.

Pathogenesis of glaucoma: Extracellular matrix dysfunction in the trabecular meshwork-A review.

The trabecular meshwork regulates aqueous humour outflow from the anterior chamber of the eye. It does this by establishing a tunable outflow resistance, defined by the interplay between cells and their extracellular matrix (ECM) milieu, and the molecular interactions between ECM proteins. During normal tissue homeostasis, the ECM is remodelled and trabecular cell behaviour is modified, permitting increased aqueous fluid outflow to maintain intraocular pressure (IOP) within a relatively narrow physiological pressure. Dysfunction in the normal homeostatic process leads to increased outflow resistance and elevated IOP, which is a primary risk factor for glaucoma. This review delineates some of the changes in the ECM that lead to gross as well as some more subtle changes in the structure and function of the ECM, and their impact on trabecular cell behaviour. These changes are discussed in the context of outflow resistance and glaucoma.

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.

Fibronectin in the Tumor Microenvironment Activates a TLR4-dependent Inflammatory Response in Lung Cancer Cells.

The microenvironment of solid tumors plays an essential role in tumor progression. In lung cancer, the stromal cells produce a fibronectin rich extracellular matrix which is known to contribute to both tumor metastasis and drug resistance. Due to its conformational lability, fibronectin is considerably remodeled by the contractile forces of the fibrotic microenvironment within the tumor stroma. As a result, the secondary structure of fibronectin's Type III domains is disrupted and the molecule becomes highly stretched. The contribution/impact of these strained forms of fibronectin on tumor growth and metastasis is not known. In the current study we show that the partially unfolded first Type III domain of fibronectin, III-1c, activates a toll-receptor/NF-κB pathway leading to an increase in the expression of IL-8. Using a 3-D model of tumor-associated extracellular matrix, we demonstrate that lung cancer cells seeded onto this matrix activate a TLR4/NF-κB signaling pathway leading to a robust increase in the release of IL-8. Cytokine release by these cells is completely dependent on the presence of fibronectin in the extracellular matrix. These findings suggest that paracrine signaling between the tumor and the stromal myofibroblasts causes a remodeling of the matrix fibronectin into a strained conformation which supports the activation of a TLR4/NF-κB signaling pathway resulting in the upregulation of fibro-inflammatory cytokines.

α5ß1 Integrin Promotes Anchoring and Integration of Transplanted Stem Cells to the Trabecular Meshwork in the Eye for Regeneration.

Stem cell-based therapy to restore the function of abnormal trabecular meshwork (TM) and decrease intraocular pressure (IOP) provides a novel approach to treat open-angle glaucoma. However, molecular mechanism for stem cells homing and anchoring to the TM remains unclear. This study aimed to discover the function of integrins in homing and integration of exogenous TM stem cells (TMSCs) to the TM. Integrin expression in TMSCs and fibroblasts was evaluated by quantitative real-time PCR (qPCR), flow cytometry, immunofluorescent staining, and western blotting. Expression of integrin ligand fibronectin was detected in cultured TM cells and murine TM tissue by immunostaining. Cell affinity to TM cells or fibronectin matrix was examined to compare TMSCs with TMSCs functionally blocked with an α5ß1 integrin antibody. TMSCs and TMSCs with α5ß1 integrin-blocking were intracamerally injected into wild-type mice. Wholemounts and cryosections were analyzed to discover cell distribution and integration at 3 days and 1 month. IOP was measured to detect possible changes. We discovered that human TMSCs expressed a higher level of α5ß1 integrin than fibroblasts, but similar levels of αvß3 and αvß5 integrin. Upregulation of fibronectin was found in both TM cells treated with dexamethasone for 14 days and murine TM tissues damaged by laser photocoagulation. TMSCs were able to attach to the TM cells and fibronectin matrix in vitro. When the surface α5ß1 integrin was blocked, the attached cell numbers were significantly reduced. Both TMSCs and TMSCs incubated with an α5ß1 integrin-blocking antibody could home to the mouse TM after injection. TMSCs blocked with the α5ß1 integrin-blocking antibody were not retained in the TM tissue at 1 month. The injected cells did not affect mouse IOP. In conclusion, highly expressed α5ß1 integrin participates in maintaining TMSCs anchored and integrated to the TM, which would be crucial for stem cell-based therapy for glaucoma.

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.

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.

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.

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.

PEGylated pUR4/FUD peptide inhibitor of fibronectin fibrillogenesis decreases fibrosis in murine Unilateral Ureteral Obstruction model of kidney disease.

Fibronectin is a blood and extracellular matrix glycoprotein that plays important roles in wound healing and fibrosis since it controls the deposition of collagen and other extracellular matrix molecules and is a substrate for infiltrating lymphocytes. Using a high-affinity fibronectin-binding peptide (FUD/pUR4) that inhibits fibronectin deposition into extracellular matrix (ECM), we tested the ability of a PEGylated FUD/pUR4 (PEG-FUD) to inhibit fibrosis in the Unilateral Ureteral Obstruction (UUO) kidney disease model. Fibronectin fibrillogenesis assays, using human fibroblasts and human proximal tubular epithelial cultures, showed that PEG-FUD can inhibit fibronectin fibrillogenesis in vitro with an IC50 similar to unconjugated FUD, in the order of 20-35 nM. In contrast, a mutated FUD (mFUD) conjugated to PEG that lacked activity did not inhibit fibronectin assembly, even at 20 µM. The in vivo activity of PEG-FUD was tested in the murine UUO model by daily subcutaneous injection of 12.5 mg/kg for 7 days until harvest at day 10. Control treatments included saline, PEG, unconjugated FUD, and PEG-mFUD. Immunoblotting studies showed that fibronectin was enriched in the extracellular matrix fractions of extracted UUO kidneys, compared to contralateral untreated kidneys. In vivo, PEG-FUD significantly decreased fibronectin by ~70% in UUO kidneys as determined by both IHC and immunoblotting, respectively. In contrast, neither PEG-mFUD, PEG, nor saline had any significant effect. PEG-FUD also decreased collagens I and III and CD45-expressing cells (leukocytes) by ~60% and ~50%, as ascertained by picrosirius red staining and IHC, respectively. Immunoblotting studies also showed that the fibronectin remaining after PEG-FUD treatment was intact. Utilizing a custom-made polyclonal antibody generated against pUR4/FUD, intact PEG-FUD was detected by immunoblotting in both the ECM and lysate fractions of UUO kidneys. No adverse reaction or event was noted with any treatment. In summary, these studies suggest that PEG-FUD reached the kidneys without degradation, and decreased fibronectin incorporation into interstitial tissue. Decreased fibronectin was accompanied by a decrease in collagen and leukocyte infiltration. We propose that PEG-FUD, a specific inhibitor of fibronectin assembly, may be a candidate therapeutic for the treatment of fibrosis in kidney diseases.

Characterization of the PEGylated Functional Upstream Domain Peptide (PEG-FUD): a Potent Fibronectin Assembly Inhibitor with Potential as an Anti-Fibrotic Therapeutic.

PURPOSE: To develop PEGylated variants of pUR4/FUD (FUD), a fibronectin assembly inhibitor, using 10 kDa, 20 kDa, and 40 kDa PEGs to evaluate their binding affinity and inhibitory potency. METHODS: The FUD peptide was recombinantly expressed, purified, and PEGylated at the N-terminus using 10 kDa, 20 kDa, and 40 kDa methoxy-PEG aldehyde. The PEGylates were purified and fractionated using ion-exchange chromatography. The molecular weight and degree of PEGylation of each conjugate was verified using MALDI-TOF. The binding affinity of each PEG-FUD conjugate was studied using isothermal titration colorimetry (ITC) and their inhibitory potency was characterized by a cell-based matrix assembly in vitro assay. RESULTS: The 10 kDa, 20 kDa, and 40 kDa PEG-FUD conjugates were synthesized and isolated in good purity as determined by HPLC analysis. Their molecular weight was consistent with attachment of a single PEG molecule to one FUD peptide. The binding affinity (Kd) and the fibronectin fibrillogenesis inhibitory potency (IC50) of all PEG-FUD conjugates remained nanomolar and unaffected by the addition of PEG. CONCLUSIONS: Retention of FUD fibronectin binding activity following PEGylation with three different PEG sizes suggest that PEG-FUD holds promise as an effective anti-fibrotic with therapeutic potential and a candidate for further pharmacokinetic and biodistribution studies.

Disruption of fibronectin matrix affects type IV collagen, fibrillin and laminin deposition into extracellular matrix of human trabecular meshwork (HTM) cells.

Fibronectin fibrils are a major component of the extracellular matrix (ECM) of the trabecular meshwork (TM). They are a key mediator of the formation of the ECM which controls aqueous humor outflow and contributes to the pathogenesis of glaucoma. The purpose of this work was to determine if a fibronectin-binding peptide called FUD, derived from the Streptococcus pyogenes Functional Upstream Domain of the F1 adhesin protein, could be used to control fibronectin fibrillogenesis and hence ECM formation under conditions where its expression was induced by treatment with the glucocorticoid dexamethasone. FUD was very effective at preventing fibronectin fibrillogenesis in the presence or absence of steroid treatment as well as the removal of existing fibronectin fibrils. Disruption of fibronectin fibrillogenesis by FUD also disrupted the incorporation of type IV collagen, laminin and fibrillin into the ECM. The effect of FUD on these other protein matrices, however, was found to be dependent upon the maturity of the ECM when FUD was added. FUD effectively disrupted the incorporation of these other proteins into matrices when added to newly confluent cells that were forming a nascent ECM. In contrast, FUD had no effect on these other protein matrices if the cell cultures already possessed a pre-formed, mature ECM. Our studies indicate that FUD can be used to control fibronectin fibrillogenesis and that these fibrils play a role in regulating the assembly of other ECM protein into matrices involving type IV collagen, laminin, and fibrillin within the TM. This suggests that under in vivo conditions, FUD would selectively disrupt fibronectin fibrils and de novo assembly of other proteins into the ECM. Finally, our studies suggest that targeting fibronectin fibril assembly may be a viable treatment for POAG as well as other glaucomas involving excessive or abnormal matrix deposition of the ECM.

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.

Involvement of Tiam1, RhoG and ELMO2/ILK in Rac1-mediated phagocytosis in human trabecular meshwork cells.

We previously demonstrated that an αvß5 integrin/FAK- mediated pathway regulated the phagocytic properties of human trabecular meshwork (HTM) cells. Here we demonstrate that this process is mediated by Rac-1 and a previously unreported signaling pathway that utilizes the Tiam1 as well as a novel ILK/RhoG/ELMO2 signaling pathway. Phagocytosis in both a TM-1 cell line and normal HTM cells was mediated by Rac1 and could be significantly decreased by >75% using the Rac1 inhibitor EHop-016. Knockdown of Rac1 in TM-1 cells also inhibited phagocytosis by 40% whereas overexpression of a constitutively active Rac1 or stimulation with PDGF increased phagocytosis by 83% and 32% respectively. Tiam1 was involved in regulating phagocytosis. Knockdown of Tiam1 inhibited phagocytosis by 72% while overexpression of Tiam1 C1199 increased phagocytosis by 75%. Other upstream effectors of Rac1 found to be involved included ELMO2, RhoG, and ILK. Knockdowns of ELMO2, ILK, and RhoG caused a reduction in phagocytosis by 51%, 55% and 46% respectively. In contrast, knockdown of Vav2 and Dock1 or overexpression of Vav2 Y159/172F did not cause a significant change in phagocytosis. These data suggest a novel link between Tiam1 and RhoG/ILK /ELMO2 pathway as upstream effectors of the Rac1-mediated phagocytic process in TM cells.

Glaucomatous MYOC mutations activate the IL-1/NF-κB inflammatory stress response and the glaucoma marker SELE in trabecular meshwork cells.

PURPOSE: Activation of the IL-1/NF-κB inflammatory stress pathway and induction of SELE expression in the trabecular meshwork (TBM) is a marker for high-tension glaucomas of diverse etiology. Pathway activation stimulates aqueous outflow and protects against oxidative stress, but may be damaging in the long-term. MYOC mutations have been causally linked to high-tension forms of primary open angle glaucoma (POAG). This study investigated a possible link between MYOC mutations and activation of the IL-1/NF-κB pathway and expression of SELE. METHODS: We constructed MYOC expression vectors with mutations at sites that cause POAG. Mutations (Q368X, Y437H, A427T) were selected to represent proteins with differing POAG-causing potency (Q368X > Y437H > A427T) and intracellular retention behavior (Q368X and Y437H retained, A427T released). The constructs were made in two different kinds of vectors; one a plasmid designed for transient transfection (pCMV6), and one a doxycycline-inducible lentiviral vector (pSLIK) for stable cell transduction. The immortalized human trabecular meshwork line TM-1 was used for all expression studies. Expression of IL1A mRNA was determined by reverse transcription (RT)-PCR, as well as a set of five other genes associated with signaling pathways linked to glaucoma: IL1B and IL6 (NF-κB pathway), TGFB2 and ACTA2 (TGF-ß pathway) and FOXO1 (E2F1 apoptotic pathway). An ELISA was used to quantify IL1A protein released into culture media. To quantify intracellular NF-κB activity, we transiently transfected stably transduced cell lines with a luciferase expression vector under control of the IL8 promoter (containing an NF-κB response element). RESULTS: Transiently expressed wild-type MYOC was released into cell culture media, whereas mutant MYOCs Q368X and Y437H remained within cells. Both mutant MYOCs activated the IL-1/ NF-κB pathway, significantly stimulating expression of IL1A and IL1B. However Y437H, which causes a severe glaucoma phenotype, was less effective than Q368X, which causes a moderate glaucoma phenotype. In addition, the retained mutants stimulated expression of stress response genes ACTA2 and FOXO1. Unexpectedly, wild-type MYOC significantly decreased expression of IL6 and TGFB2, to approximately half of the control levels, and expression of IL1B and ACTA2 was also slightly decreased. Induction of MYOC mutants Q368X and Y437H in stably transduced cell lines significantly stimulated the level of IL1A protein released into culture media. Once again however, the effect of the severe MYOC mutant Y437H was less than the effect of the moderate MYOC mutant Q368X. In contrast, induced expression of the intracellularly retained mutant MYOC A427T or wild-type MYOC did not change the amount of IL1A protein in culture media. Induction of Y437H MYOC plus IL1A treatment increased NF-κB activity by 25% over IL1A alone. In contrast, induction of Q368X or A427T plus IL1A treatment did not significantly affect NF-κB activity over IL1A alone. However, wild-type MYOC expression inhibited IL1A-stimulated NF-κB activity. We also observed that endogenous MYOC expression was induced by IL1A in TM-1 cells and primary TBM cell cultures. SELE was co-expressed with MYOC in the primary cell lines. CONCLUSIONS: These results indicate that POAG-causing MYOC mutants activate the IL-1/NF-κB pathway, with activation levels correlated with intracellular retention of the protein, but not POAG-causing potency. Unexpectedly, it was also discovered that wild-type MYOC inhibits activation of the IL-1/NF-κB pathway, and that activation of the IL-1/NF-κB pathway stimulates expression of MYOC. This is the first evidence that glaucoma-causing MYOC mutants can activate the inflammatory response and that wild-type MYOC has anti-inflammatory activity.