Development of an in vitro model to study the biological effects of blinking.

PURPOSE: To develop a mechanical model in which a contact lens is swept over ocular surface cells under conditions that mimic the force and speed of the blink, and to investigate the resulting biological changes. METHODS: A computer controlled mechanical instrument was developed to hold a dish containing 3D cultured stratified human ocular surface epithelial cells, across which an arm bearing a contact lens was swept back and forth repeatedly at a speed and force mimicking the human blink. Cells were subjected to repeated sweep cycles for up to 1 h at a speed of 120 mm/s with or without an applied force of 19.6 mN (to mimic pressure exerted by upper eyelid), after which the cell layer thickness was measured, the cell layer integrity was investigated using fluorescent quantum dots (6 and 13 nm) and the phosphorylation levels of various protein kinases were analyzed by human phospho-kinase arrays. Data for selected kinases were further quantitated by enzyme immunoassays. RESULTS: The thickness of the cell layers did not change after exposure to sweep cycles with or without applied force. Quantum dots (6 and 13 nm) were able to penetrate the layers of cells exposed to sweep cycles but not layers of untreated control cells. The phosphorylation levels of HSP27 and JNK1/2/3 increased for cells exposed to sweep cycles with applied force compared to untreated control cells. CONCLUSIONS: The in vitro mechanical instrument is a useful tool to investigate the effects of blinking on the ocular surface.

Consequences of Preservative Uptake and Release by Contact Lenses.

OBJECTIVES: To assess contact lens preservative uptake and release from multipurpose solutions (MPS) and subsequent acquisition of lens antibacterial activity. METHODS: Kinetics of uptake and release of poly (hexamethylene biguanide hydrochloride) (PHMB) or polyquaternium-1 (PQ-1) from various contact lenses were studied initially with the pure compounds and then after soaking in MPS containing these compounds. Lenses soaked in MPS were tested for antibacterial activity. RESULTS: Only lenses with a negatively charged component absorbed these preservatives. For lenses containing methacrylic acid (MA), uptake of PHMB from preservative-only solution was fast, yet little was released, in contrast to its rapid release from lenses containing other anionic groups. This trend persisted with PHMB-containing MPS. PQ-1 from preservative-only solution was only absorbed by lenses containing MA and was released from MA-containing hydrogels, but not significantly from an MA-containing silicone hydrogel. Lens uptake of PQ-1 was much lower from MPS and release was essentially undetectable from all lenses evaluated. Antibacterial lens activity was acquired by lenses containing MA after an overnight soak in MPS containing PQ-1, and for balafilcon A and omafilcon A after 5 exchanges in PHMB-containing MPS. Acquired activity was maintained during cycling between artificial tear protein solution and MPS. CONCLUSIONS: Lens preservative uptake and its subsequent release are dependent on lens chemistry, preservative nature, and other MPS components. A few lens/solution combinations acquired antibacterial activity after one or more overnight soaks in MPS, depending on the nature of the anionic lens component and the preservative. Uncharged lenses did not acquire antibacterial activity.

Effects of MMP12 on cell motility and inflammation during corneal epithelial repair.

Corneal epithelial defects are a common cause of ocular morbidity and can result in corneal scarring if they do not heal properly. Matrix metalloproteinases (MMPs) are extracellular matrix proteinases that regulate multiple aspects of corneal repair. We have previously shown that MMP12 has a protective effect on corneal fibrosis through its regulation of neutrophil and macrophage infiltration and angiogenesis in a chemical injury model involving full thickness damage to the cornea. However, the role of MMP12 in injuries limited to the corneal epithelium is relatively unknown. This study investigates the reparative effects of MMP12 following isolated corneal epithelial injury. Using a corneal epithelial debridement injury model performed on corneas of wild-type (WT) mice, we show that Mmp12 is expressed early following corneal epithelial injury with highest expression levels at 8 h after injury and lower expression levels at 4 and 8 days after injury. We investigated whether MMP12 has an effect on the rate of epithelial repair and cell migration using in vivo and in vitro scratch assays performed on WT and Mmp12-/- mice. We found that loss of MMP12 results in a slower scratch wound repair rate both in vivo and in vitro. We also found that corneas of Mmp12-/- mice have decreased neutrophil infiltration following injury. Loss of MMP12, however, does not affect cell proliferation in the center of the wounds. These data support a role of MMP12 in promoting early repair processes following corneal epithelial injury by enhancing epithelial cell migration and neutrophil infiltration.

The SULFs, extracellular sulfatases for heparan sulfate, promote the migration of corneal epithelial cells during wound repair.

Corneal epithelial wound repair involves the migration of epithelial cells to cover the defect followed by the proliferation of the cells to restore thickness. Heparan sulfate proteoglycans (HSPGs) are ubiquitous extracellular molecules that bind to a plethora of growth factors, cytokines, and morphogens and thereby regulate their signaling functions. Ligand binding by HS chains depends on the pattern of four sulfation modifications, one of which is 6-O-sulfation of glucosamine (6OS). SULF1 and SULF2 are highly homologous, extracellular endosulfatases, which post-synthetically edit the sulfation status of HS by removing 6OS from intact chains. The SULFs thereby modulate multiple signaling pathways including the augmentation of Wnt/ß-catenin signaling. We found that wounding of mouse corneal epithelium stimulated SULF1 expression in superficial epithelial cells proximal to the wound edge. Sulf1⁻/⁻, but not Sulf2⁻/⁻, mice, exhibited a marked delay in healing. Furthermore, corneal epithelial cells derived from Sulf1⁻/⁻ mice exhibited a reduced rate of migration in repair of a scratched monolayer compared to wild-type cells. In contrast, human primary corneal epithelial cells expressed SULF2, as did a human corneal epithelial cell line (THCE). Knockdown of SULF2 in THCE cells also slowed migration, which was restored by overexpression of either mouse SULF2 or human SULF1. The interchangeability of the two SULFs establishes their capacity for functional redundancy. Knockdown of SULF2 decreased Wnt/ß-catenin signaling in THCE cells. Extracellular antagonists of Wnt signaling reduced migration of THCE cells. However in SULF2- knockdown cells, these antagonists exerted no further effects on migration, consistent with the SULF functioning as an upstream regulator of Wnt signaling. Further understanding of the mechanistic action of the SULFs in promoting corneal repair may lead to new therapeutic approaches for the treatment of corneal injuries.

Protective effects of matrix metalloproteinase-12 following corneal injury.

Corneal scarring due to injury is a leading cause of blindness worldwide and results from dysregulated inflammation and angiogenesis during wound healing. Here we demonstrate that the extracellular matrix metalloproteinase MMP12 (macrophage metalloelastase) is an important regulator of these repair processes. Chemical injury resulted in higher expression of the fibrotic markers α-smooth muscle actin and type I collagen, and increased levels of angiogenesis in corneas of Mmp12(-/-) mice compared with corneas of wild-type mice. In vivo, we observed altered immune cell dynamics in Mmp12(-/-) corneas by confocal imaging. We determined that the altered dynamics were the result of an altered inflammatory response, with delayed neutrophil infiltration during the first day and excessive macrophage infiltration 6 days later, mediated by altered expression levels of chemokines CXCL1 and CCL2, respectively. Corneal repair returned to normal upon inhibition of these chemokines. Taken together, these data show that MMP12 has a protective effect on corneal fibrosis during wound repair through regulation of immune cell infiltration and angiogenesis.

AsialoGM1 and TLR5 cooperate in flagellin-induced nucleotide signaling to activate Erk1/2.

Bacterial flagellin can interact with both Toll-like receptor 5 (TLR5) and the cell surface glycolipid, asialoGM1, to activate an innate immune response. The induction of mucin by flagellin in human lung epithelial cells (NCIH292) is dependent on asialoGM1 ligation, ATP receptor signaling, Ca2+ mobilization, and Erk1/2 activation. Conversely, the activation of NF-kappaB by flagellin is dependent on signaling through TLR5. These results prompted us to ask whether the flagellin-induced TLR5 signaling pathway was intersecting with or mutually independent of the nucleotide receptor pathway activated downstream of asialoGM1. Herein, we demonstrate that the release of ATP induced by flagellin is dependent on a Toll signaling cascade. Although Toll was able to activate NF-kappaB in the absence of extracellular ATP, Toll required ATP to activate Erk1/2. These results suggest interdependence between the asialoGM1 and TLR5 pathways and reveal a previously unsuspected role for autocrine extracellular ATP signaling in TLR signaling.

Adenosine up-regulation of the mucin gene, MUC2, in asthma.

Mucus hypersecretion is a hallmark of asthma that contributes to airway obstruction. While the etiology is not well understood, hypersecretion has been linked to the presence of cytokines such as IL-4, IL-5, IL-9, and IL-13 in the inflamed airway. The presence of adenosine has also been noted in asthmatic airways, and adenosine-mediated signaling in mast cells has been implicated in the severe bronchoconstriction and inflammation prevalent in these patients (1, 2). Here we examine the possibility that adenosine also contributes to mucus hypersecretion by airway epithelial cells. Results in cultured airway epithelial cells showed that MUC2 mucin expression increased in response to adenosine. This appeared to be mediated by a pathway initiated at the adenosine A1 receptor that transduced signals through a Ca2+-activated Cl- channel and EGFR. That this signaling cascade is relevant to asthmatic hypersecretion was indicated by results showing that mucin induction by asthmatic tracheal aspirates was reduced by A1, CLCA1, and EGFR inhibitors. These results suggest that adenosine cooperates with inflammatory cytokines to stimulate mucin production in the asthmatic airway and supports the use of A1, CLCA1, and EGFR inhibitors in the treatment of asthma.

Pseudomonas aeruginosa internalization by corneal epithelial cells involves MEK and ERK signal transduction proteins.

Invasion of epithelial cells represents a potential pathogenic mechanism for Pseudomonas aeruginosa. We explored the role of mitogen-activated protein kinase kinases (MEK 1/2) and the extracellular signal-regulated kinases (ERK 1/2) in P. aeruginosa invasion. Treatment of corneal epithelial cells with MEK inhibitors, PD98059 (20 microM) or UO126 (100 microM), reduced P. aeruginosa invasion by approximately 60% without affecting bacterial association with the cells (P=0.0001). UO124, a negative control for UO126, had no effect on bacterial internalization. Infection of cells with an internalization-defective flhA mutant of P. aeruginosa was associated with less ERK 1/2 tyrosine phosphorylation than infection with wild-type invasive P. aeruginosa. An ERK-2 inhibitor, 5-iodotubercidin (20 microM), reduced P. aeruginosa invasion by approximately 40% (P=0.035). Together, these data suggest that P. aeruginosa internalization by epithelial cells involves a pathway(s) that includes MEK and ERK signaling proteins.