Insulin-like growth factor binding protein-3 mediates hyperosmolar stress-induced mitophagy through the mechanistic target of rapamycin.

Hyperosmolarity of the ocular surface triggers inflammation and pathological damage in dry eye disease (DED). In addition to a reduction in quality of life, DED causes vision loss and when severe, blindness. Mitochondrial dysfunction occurs as a consequence of hyperosmolar stress. We have previously reported on a role for the insulin-like growth factor binding protein-3 (IGFBP-3) in the regulation of mitochondrial ultrastructure and metabolism in mucosal surface epithelial cells; however, this appears to be context-specific. Due to the finding that IGFBP-3 expression is decreased in response to hyperosmolar stress in vitro and in an animal model of DED, we next sought to determine whether the hyperosmolar stress-mediated decrease in IGFBP-3 alters mitophagy, a key mitochondrial quality control mechanism. Here we show that hyperosmolar stress induces mitophagy through differential regulation of BNIP3L/NIX and PINK1-mediated pathways. In corneal epithelial cells, this was independent of p62. The addition of exogenous IGFBP-3 abrogated the increase in mitophagy. This occurred through regulation of mTOR, highlighting the existence of a new IGFBP-3-mTOR signaling pathway. Together, these findings support a novel role for IGFBP-3 in mediating mitochondrial quality control in DED and have broad implications for epithelial tissues subject to hyperosmolar stress and other mitochondrial diseases.

Extracellular vesicles released by host epithelial cells during Pseudomonas aeruginosa infection function as homing beacons for neutrophils.

BACKGROUND: Pseudomonas aeruginosa (PA) is an opportunistic pathogen that can cause sight threatening infections in the eye and fatal infections in the cystic fibrosis airway. Extracellular vesicles (EVs) are released by host cells during infection and by the bacteria themselves; however, there are no studies on the composition and functional role of host-derived EVs during PA infection of the eye or lung. Here we investigated the composition and capacity of EVs released by PA infected epithelial cells to modulate innate immune responses in host cells. METHODS: Human telomerase immortalized corneal epithelial cells (hTCEpi) cells and human telomerase immortalized bronchial epithelial cells (HBECs) were treated with a standard invasive test strain of Pseudomonas aeruginosa, PAO1, for 6 h. Host derived EVs were isolated by qEV size exclusion chromatography. EV proteomic profiles during infection were compared using mass spectrometry and functional studies were carried out using hTCEpi cells, HBECs, differentiated neutrophil-like HL-60 cells, and primary human neutrophils isolated from peripheral blood. RESULTS: EVs released from PA infected corneal epithelial cells increased pro-inflammatory cytokine production in naïve corneal epithelial cells and induced neutrophil chemotaxis independent of cytokine production. The EVs released from PA infected bronchial epithelial cells were also chemotactic although they failed to induce cytokine secretion from naïve HBECs. At the proteomic level, EVs derived from PA infected corneal epithelial cells exhibited lower complexity compared to bronchial epithelial cells, with the latter having reduced protein expression compared to the non-infected control. CONCLUSIONS: This is the first study to comprehensively profile EVs released by corneal and bronchial epithelial cells during Pseudomonas infection. Together, these findings show that EVs released by PA infected corneal and bronchial epithelial cells function as potent mediators of neutrophil migration, contributing to the exuberant neutrophil response that occurs during infection in these tissues.

Pseudomonas aeruginosa-Derived Extracellular Vesicles Modulate Corneal Inflammation: Role in Microbial Keratitis?

Pseudomonas aeruginosa keratitis occurs following trauma, in immunocompromised patients, and in otherwise healthy contact lens wearers. Characterized by a light-blocking infiltrate, P. aeruginosa keratitis is the most serious complication associated with contact lens wear and, in severe cases, can lead to vision loss. Bacterial extracellular vesicles (B EVs) are membrane-enclosed nanometer-scale particles secreted from bacteria and are packed with bioactive molecules. B EVs have been shown to mediate biological functions that regulate host pathogenic responses. In the present study, we isolated P. aeruginosa-derived EVs using size exclusion chromatography and compared the proteomic compositions and functional activities of P. aeruginosa-derived EVs and P. aeruginosa-derived free protein (FP) on corneal epithelial cells and neutrophils. Importantly, P. aeruginosa-derived EVs and FP exhibited unique protein profiles, with EVs being enriched in P. aeruginosa virulence proteins. P. aeruginosa-derived EVs promoted corneal epithelial cell secretion of interleukin-6 (IL-6) and IL-8, whereas these cytokines were not upregulated following treatment with FP. In contrast, FP had a negative effect on the host inflammatory response and impaired neutrophil killing. Both P. aeruginosa-derived EVs and FP promoted intracellular bacterial survival in corneal epithelial cells. Collectively, these data suggest that P. aeruginosa-derived EVs and FP may play a critical role in the pathogenesis of corneal infection by interfering with host innate immune defense mechanisms.

IGFBP-3 functions as a molecular switch that mediates mitochondrial and metabolic homeostasis.

Mitochondrial dysfunction or loss of homeostasis is a central hallmark of many human diseases. Mitochondrial homeostasis is mediated by multiple quality control mechanisms including mitophagy, a form of selective autophagy that recycles terminally ill or dysfunctional mitochondria in order to preserve mitochondrial integrity. Our prior studies have shown that members of the insulin-like growth factor (IGF) family localize to the mitochondria and may play important roles in mediating mitochondrial health in the corneal epithelium, an integral tissue that is required for the maintenance of optical transparency and vision. Importantly, the IGF-binding protein-3, IGFBP-3, is secreted by corneal epithelial cells in response to stress and functions to mediate intracellular receptor trafficking in this cell type. In this study, we demonstrate a novel role for IGFBP-3 in mitochondrial homeostasis through regulation of the short isoform (s)BNIP3L/NIX mitophagy receptor in corneal epithelial cells and extend this finding to non-ocular epithelial cells. We further show that IGFBP-3-mediated control of mitochondrial homeostasis is associated with alterations in lamellar cristae morphology and mitochondrial dynamics. Interestingly, both loss and gain of function of IGFBP-3 drive an increase in mitochondrial respiration. This increase in respiration is associated with nuclear accumulation of IGFBP-3. Taken together, these findings support a novel role for IGFBP-3 as a key mediator of mitochondrial health in mucosal epithelia through the regulation of mitophagy and mitochondrial morphology.

Temporal and Spatial Assessment of the Corneal Response to UV Cross-Linking Using 3-Dimensional In Vivo Confocal Microscopy.

ABSTRACT: The goal of this study was to evaluate the temporal and spatial pattern of wound healing following UV corneal cross-linking (CXL) using 3-dimensional (3-D) confocal imaging in vivo. Using a modified Heidelberg Retinal Tomograph with Rostock Corneal Module confocal microscope, we performed 3-D scans on two patients at multiple time points after CXL. Patient 1 showed a normal post-CXL wound healing response, with initial subbasal nerve loss and keratocyte apoptosis in the anterior stroma, followed by partial restoration of both the nerve plexus and stromal keratocytes by 6 months. In patient 2, in addition to anterior corneal damage, pyknotic nuclei were observed in the posterior stroma 7 days after CXL. Acellular areas were present in the posterior stroma at 3 months, with only partial keratocyte repopulation at 6 months. Regeneration of the subbasal nerve plexus was also delayed. Three-dimensional confocal imaging allowed these unusual wound healing responses to be identified in the absence of any corresponding clinical observations.

IGFBP-3 Regulates Mitochondrial Hyperfusion and Metabolic Activity in Ocular Surface Epithelia during Hyperosmolar Stress.

In the eye, hyperosmolarity of the precorneal tear film triggers inflammation and the development of dry eye disease (DED), a highly prevalent condition that causes depression and disability in severe forms. A member of the insulin-like growth factor (IGF) family, the IGF binding protein-3 (IGFBP-3), is a pleiotropic protein with known roles in growth downregulation and survival. IGFBP-3 exerts these effects by blocking IGF-1 activation of the type 1 IGF-receptor (IGF-1R). Here, we examined a new IGF-independent role for IGFBP-3 in the regulation of mitochondrial and metabolic activity in ocular surface epithelial cells subject to hyperosmolar stress and in a mouse model of DED. We found that hyperosmolar stress decreased IGFBP-3 expression in vitro and in vivo. Treatment with exogenous IGFBP-3 induced an early, transient shift in IGF-1R to mitochondria, followed by IGFBP-3 nuclear accumulation. IGFBP-3 nuclear accumulation increased protein translation, blocked the hyperosmolar-mediated decrease in oxidative phosphorylation through the induction of mitochondrial hyperfusion, and restored corneal health in vivo. These data indicate that IGFBP-3 acts a stress response protein in ocular surface epithelia subject to hyperosmolar stress. These findings may lead to the development of first-in-class therapeutics to treat eye diseases with underlying mitochondrial dysfunction.

Insulin receptor preserves mitochondrial function by binding VDAC1 in insulin insensitive mucosal epithelial cells.

Unlike many epithelial tissues, the corneal epithelium is insulin insensitive, meaning it does not require insulin for glucose uptake. In this study, we show that insulin differentially regulates mitochondrial respiration in two human mucosal epithelial cell types: insulin-insensitive corneal epithelial cells and insulin-sensitive bronchial epithelial cells. In both cell types, insulin blocks glycogen synthase kinase beta (GSK3ß) activity. In the corneal epithelium however, insulin selectively regulates PTEN-induced kinase 1 (PINK-1)-mediated mitophagy and mitochondrial accumulation of insulin receptor (INSR). While insulin blocked basal levels of PINK-1-mediated mitophagy in bronchial epithelial cells, mitochondrial trafficking of INSR was not detectable. We further show that in corneal epithelia, INSR interacts with the voltage-dependent anion channel-1 (VDAC1) in mitochondria and that INSR knockdown triggers robust mitochondrial fragmentation, alterations in mitochondrial polarization, and blocks the induction of PINK-1-mediated mitophagy. Collectively, these data demonstrate that INSR interacts with VDAC1 to mediate mitochondrial stability. We also demonstrate unique interactions between VDAC1 and other receptor tyrosine kinases, indicating a novel role for this family of receptors in mitochondria.

Tear Levels of IGFBP-3: A Potential Biomarker for Diabetic Nerve Changes in the Cornea.

INTRODUCTION: Type 2 diabetes mellitus has reached epidemic levels in the United States and worldwide. Ocular complications from this disease include diabetic retinopathy and keratopathy, both of which can lead to significant vision loss. While frequently underappreciated, diabetic keratopathy is associated with painful ocular surface disorders, including corneal erosions and delayed wound healing. Recent work in our laboratory has focused on the role of the insulin-like growth factor (IGF) system in diabetic corneal disease. METHODS: Here, we review recent findings on the presence of IGF-1, insulin, and the insulin-like binding protein (IGFBP-3) in human tear fluid and evaluate their potential use as biomarkers in diabetes. We further examine clinical evidence using in vivo confocal microscopy as an important imaging biomarker in diabetes and discuss associations between tear film changes in diabetes and corneal nerve loss. RESULTS: IGFBP-3 was the only tear film marker significantly associated with nerve loss in type 2 diabetes, whereas tear levels of IGF-1 were associated with aging. Interestingly, tear levels of IGFBP-3 were not directly related to serum levels of HbA1c, suggesting that hyperglycemia alone is not driving increased secretion of this protein. CONCLUSIONS: Overwhelming evidence supports the use of in vivo confocal microscopy as a tool to evaluate corneal nerve and epithelial changes induced by diabetes in research settings. The newly identified relationship between morphological changes in the corneal subbasal nerve plexus in diabetes and the increase in tear levels of IGFBP-3 suggest that this protein may represent an innovative new biomarker to assess risk of ocular and nonocular complications in type 2 diabetes mellitus.

Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells.

The insulin-like growth factor type 1 receptor (IGF-1R) is part of the receptor tyrosine kinase superfamily. The activation of IGF-1R regulates several key signaling pathways responsible for maintaining cellular homeostasis, including survival, growth, and proliferation. In addition to mediating signal transduction at the plasma membrane, in serum-based models, IGF-1R undergoes SUMOylation by SUMO 1 and translocates to the nucleus in response to IGF-1. In corneal epithelial cells grown in serum-free culture, however, IGF-1R has been shown to accumulate in the nucleus independent of IGF-1. In this study, we report that the insulin-like growth factor binding protein-3 (IGFBP-3) mediates nuclear translocation of IGF-1R in response to growth factor withdrawal. This occurs via SUMOylation by SUMO 2/3. Further, IGF-1R and IGFBP-3 undergo reciprocal regulation independent of PI3k/Akt signaling. Thus, under healthy growth conditions, IGFBP-3 functions as a gatekeeper to arrest the cell cycle in G0/G1, but does not alter mitochondrial respiration in cultured cells. When stressed, IGFBP-3 functions as a caretaker to maintain levels of IGF-1R in the nucleus. These results demonstrate mutual regulation between IGF-1R and IGFBP-3 to maintain cell survival under stress. This is the first study to show a direct relationship between IGF-1R and IGFBP-3 in the maintenance of corneal epithelial homeostasis.

Acceleration of the formation of biofilms on contact lens surfaces in the presence of neutrophil-derived cellular debris is conserved across multiple genera.

Purpose: We have previously shown that invasive strains of Pseudomonas aeruginosa exploit the robust neutrophil response to form biofilms on contact lens surfaces and invade the corneal epithelium. The present study investigated the ability of multiple bacterial genera, all commonly recovered during contact lens-related infectious events, to adhere to and form biofilms on contact lens surfaces in the presence of neutrophils. Methods: Five reference strains from the American Type Culture Collection were used: P. aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia, Staphylococcus aureus, and Staphylococcus epidermidis. Each bacterial strain was incubated overnight with or without stimulated human neutrophils in the presence of an unworn contact lens. Standard colony counts and laser scanning confocal microscopy of BacLight-stained contact lenses were used to assess bacterial viability. Three-dimensional modeling of lens-associated biofilms with Imaris software was used to determine the biofilm volume. Lenses were further examined using scanning electron microscopy. Results: Less than 1% of the starting inoculum adhered to the contact lens surface incubated with bacteria alone. There were no differences in adhesion rates to contact lens surfaces between bacteria in the absence of neutrophils for either the Gram-negative or Gram-positive test strains. Bacterial adhesion to contact lens surfaces was accelerated in the presence of human neutrophils for all test strains. This effect was least evident with S. epidermidis. There was also an increase in the number of viable bacteria recovered from contact lens surfaces (p<0.001 for the Gram-negative and Gram-positive test strains, respectively) and in biofilm volume (p<0.001 for the Gram-negative test strains, p = 0.005 for S. aureus). Conclusions: These results show that in addition to P. aeruginosa, other bacteria commonly encountered during contact lens wear possess the capacity to utilize neutrophil-derived cellular debris to facilitate colonization of the lens surface. These data suggest that this phenomenon is conserved among multiple genera. Thus, during contact lens wear, the presence of inflammation and the accumulation of neutrophil debris under the posterior lens surface likely contribute to colonization of the lens. Further studies are needed to correlate these findings with risk for infection in an animal model.

The impact of type 1 diabetes mellitus on corneal epithelial nerve morphology and the corneal epithelium.

Diabetic corneal neuropathy can result in chronic, sight-threatening corneal pathology. Although the exact etiology is unknown, it is believed that a reduction in corneal sensitivity and loss of neurotrophic support contributes to corneal disease. Information regarding the relationship between nerve loss and effects on the corneal epithelium is limited. We investigated changes in the corneal epithelium and nerve morphology using three-dimensional imaging in vivo and in situ in a streptozotocin-induced diabetic mouse model. Streptozotocin-treated mice showed increased levels of serum glucose and growth retardation consistent with a severe diabetic state. A reduction in the length of the subbasal nerve plexus was evident after 6 weeks of disease. Loss of the subbasal nerve plexus was associated with corneal epithelial thinning and a reduction in basal epithelial cell density. In contrast, loss of the terminal epithelial nerves was associated with animal age. Importantly, this is the first rodent model of type 1 diabetes that shows characteristics of corneal epithelial thinning and a reduction in basal epithelial cell density, both previously have been documented in humans with diabetic corneal neuropathy. These findings indicate that in type 1 diabetes, nerve fiber damage is evident in the subbasal nerve plexus before terminal epithelial nerve loss and that neurotrophic support from both the subbasal nerve plexus and terminal epithelial nerves is essential for the maintenance of corneal epithelial homeostasis.

Cornea and ocular surface disease: application of cutting-edge optometric research.

: Clinician-scientists bridge the gap between basic research and patient care. At the 2012 Annual Meeting, a symposium highlighting the application of cutting-edge optometric research within the anterior segment was held to present and discuss some of the recent basic scientific advances that will both shape and guide the development of future clinical care practices. This article summarizes this work, bringing together four experts, all clinician-scientists in the field of cornea and ocular surface. Collectively, this work provides new insights to clinicians and researchers alike, as well as brings forth a greater appreciation of the impact of ongoing optometric bench research in advancing clinical care.

Pseudomonas aeruginosa impairs mitochondrial function and metabolism during infection of corneal epithelial cells.

Pseudomonas aeruginosa (PA) is a gram-negative opportunistic pathogen that can infect the cornea as a result of trauma or contact lens wear. In addition to their known energy producing role, mitochondria are important mediators of immune signaling and host defense. While certain pathogens have developed strategies to evade host defenses by modulating host mitochondrial dynamics and metabolism, the ability of PA to harness host cell mitochondria during corneal infection is unknown. Using a combination of biochemical and imaging techniques, we show that PA infection of corneal epithelial cells induced mitochondrial fission in a DRP1-dependent manner that preceded PINK1/Parkin and FUNDC1-mediated mitophagy. PA also impaired NADH-linked respiration through a reduction in complex 1. This corresponded to a decrease in metabolic pathways related to glycolysis and the TCA cycle. Metabolomics analysis further demonstrated an upregulation of the pentose phosphate pathway, arginine, purine, and pyrimidine metabolism in PA infected cells. These pathways may provide a key source of nucleotides, amino acids, and nitrogen for both the host cell and PA, in addition to antioxidant functions. Following treatment with gentamicin to kill all extracellular bacteria, metabolic flux analysis showed that corneal epithelial cells were able to restore mitochondrial function despite the continued presence of intracellular PA. Taken together, these data demonstrate that mitochondrial dysfunction and metabolic rewiring in host cells is triggered by extracellular PA, but once inside, PA requires healthy mitochondria to ensure host cell survival.

The effects of silicone hydrogel lens wear on the corneal epithelium and risk for microbial keratitis.

Previous studies using animal models and human clinical trials have demonstrated that the use of low-oxygen-transmissible contact lens materials produce corneal epithelial surface damage resulting in increased Pseudomonas aeruginosa (PA) adhesion and raft-mediated internalization into surface corneal epithelial cells. These findings led to the testable clinical predictions that (1) microbial keratitis (MK) risk is expected to be the greatest during the first 6 months of wear; (2) there is no difference between 6 and 30 night extended wear; and (3) that wear of hyperoxygen-transmissible lenses would reduce the reported incidence of infection. Subsequent epidemiologic studies have confirmed the first two predictions; however, increased oxygen transmissibility with silicone hydrogel (SiHy) lens wear has not altered the overall incidence of MK. In this review, more recent clinical and basic studies that investigate epithelial alterations and bacterial adhesion to corneal epithelial cells after the wear of SiHy lenses with and without concomitant exposure to chemically preserved multipurpose solutions (MPS) will be examined. The collective results of these studies demonstrate that even in the absence of lens-related hypoxia, MPS induce ocular surface changes during SiHy lens wear that are associated with a pathophysiologic increase in PA adherence and internalization in the corneal epithelium, and therefore, predict a greater risk for PA-MK. In addition, new data supporting an interactive role for inflammation in facilitating PA adherence and internalization in the corneal epithelium will also be discussed.

The roles of autophagy and mitophagy in corneal pathology: current knowledge and future perspectives.

The cornea is the clear dome that covers the front portion of the globe. The primary functions of the cornea are to promote the refraction of light and to protect the eye from invading pathogens, both of which are essential for the preservation of vision. Homeostasis of each cellular layer of the cornea requires the orchestration of multiple processes, including the ability to respond to stress. One mechanism whereby cells respond to stress is autophagy, or the process of "self-eating." Autophagy functions to clear damaged proteins and organelles. During nutrient deprivation, amino acids released from protein breakdown via autophagy are used as a fuel source. Mitophagy, a selective form of autophagy, functions to clear damaged mitochondria. Thus, autophagy and mitophagy are important intracellular degradative processes that sustain tissue homeostasis. Importantly, the inhibition or excessive activation of these processes result in deleterious effects on the cell. In the eye, impairment or inhibition of these mechanisms have been associated with corneal disease, degenerations, and dystrophies. This review summarizes the current body of knowledge on autophagy and mitophagy at all layers in the cornea in both non-infectious and infectious corneal disease, dystrophies, and degenerations. It further highlights the critical gaps in our understanding of mitochondrial dysfunction, with implications for novel therapeutics in clinical practice.

Loss of Corneal Nerves and Corneal Haze in Patients with Fuchs' Endothelial Corneal Dystrophy with the Transcription Factor 4 Gene Trinucleotide Repeat Expansion.

Objective: Seventy percent of Fuchs' endothelial corneal dystrophy (FECD) cases are caused by an intronic trinucleotide repeat expansion in the transcription factor 4 gene (TCF4). The objective of this study was to characterize the corneal subbasal nerve plexus and corneal haze in patients with FECD with (RE+) and without the trinucleotide repeat expansion (RE-) and to assess the correlation of these parameters with disease severity. Design: Cross-sectional, single-center study. Participants: Fifty-two eyes of 29 subjects with a modified Krachmer grade of FECD severity from 1 to 6 were included in the study. Fifteen of the 29 subjects carried an expanded TCF4 allele length of ≥ 40 cytosine-thymine-guanine repeats (RE+). Main Outcomes Measures: In vivo confocal microscopy assessments of corneal nerve fiber length (CNFL), corneal nerve branch density, corneal nerve fiber density (CNFD), and anterior corneal stromal backscatter (haze); Scheimpflug tomography densitometry measurements of haze in anterior, central, and posterior corneal layers. Results: Using confocal microscopy, we detected a negative correlation between FECD severity and both CNFL and CNFD in the eyes of RE+ subjects (Spearman ρ = -0.45, P = 0.029 and ρ = -0.62, P = 0.0015, respectively) but not in the eyes of RE- subjects. Additionally, CNFD negatively correlated with the repeat length of the expanded allele in the RE+ subjects (Spearman ρ = -0.42, P = 0.038). We found a positive correlation between anterior stromal backscatter and severity in both the RE+ and RE- groups (ρ = 0.60, P = 0.0023 and ρ = 0.44, P = 0.024, respectively). The anterior, central, and posterior Scheimpflug densitometry measurements also positively correlated with severity in both the RE+ and RE- groups (P = 5.5 × 10-5, 2.5 × 10-4, and 2.9 × 10-4, respectively, after adjusting for the expansion status in a pooled analysis. However, for patients with severe FECD (Krachmer grades 5 and 6), the posterior densitometry measurements were higher in the RE+ group than in the RE- group (P < 0.05). Conclusions: Loss of corneal nerves in FECD supports the classification of the TCF4 trinucleotide repeat expansion disorder as a neurodegenerative disease. Haze in the anterior, central, and posterior cornea correlate with severity, irrespective of the genotype. Quantitative assessments of corneal nerves and corneal haze may be useful to gauge and monitor FECD disease severity in RE+ patients.

Cellular distribution of the IGF-1R in corneal epithelial cells.

This study characterized the expression and subcellular localization of the IGF-1R in human corneal epithelial cells. Using a human telomerase-immortalized corneal epithelial cell line, IGF-1R expression and localization was assayed by immunofluorescence and subcellular fractionation followed by western blot. IGF-1R expression was confirmed in primary cultured human corneal epithelial cells. Nuclear localization was assessed under basal and IGF-1 stimulated culture conditions; phosphorylation status of the receptor in response to IGF-1 was demonstrated by western blot. IGF-1R:E-cadherin interactions were detected by immunofluorescence and co-immunoprecipitation of whole cell lysates. The results of this study demonstrated that IGF-1R localized predominantly to the nucleus and in a perinuclear cap pattern which co-localized with the Golgi complex in proliferating corneal epithelial cells. There was no difference in nuclear localization between primary or telomerized cell lines. Subcellular fractionation confirmed IGF-1Rα- and ß-subunit localization in soluble and chromatin-bound nuclear fractions. Neither growth factor withdrawal nor IGF-1 stimulation altered nuclear IGF-1R. At points of cell-cell contact, IGF-1R co-localized with E-cadherin; co- immunoprecipitation assays confirmed the presence of an IGF-1R:E-cadherin complex. Importantly, this is the first report to identify IGF-1R in the nucleus and complexed with E-cadherin at points of cell-cell contact in corneal epithelial cells. Nuclear trafficking appeared to be independent of ligand-mediated events at the plasma membrane. The identification of IGF-1R in the nucleus and complexed with E-cadherin suggests novel regulatory functions outside the canonical ligand-induced endocytosis signaling pathway.

Elevated IGFBP3 levels in diabetic tears: a negative regulator of IGF-1 signaling in the corneal epithelium.

To determine the ratio of IGFBP3:IGF-1 in normal and diabetic human tears, and in telomerase-immortalized human corneal epithelial cells (hTCEpi) cultured under elevated glucose conditions and to correlate these changes with total and phosphorylated levels of IGF-1R. Tear samples were collected noninvasively from diabetic subjects and non-diabetic controls; corneal sensitivity was assessed using a Cochet-Bonnet Aesthesiometer. Conditioned media were collected following culture of hTCEpi cells in normal (5 mM) and elevated (25 mM) glucose conditions; mannitol was used as an osmotic control. IGFBP3, IGF-1, and phosphorylated IGF-1R levels were assessed by ELISA. IGFBP3 and IGF-1R mRNA were assessed by real-time polymerase chain reaction (PCR). Total and phosphorylated IGF-1R expression in whole cell lysates was assessed by western blot. There was a 2.8-fold increase in IGFBP3 in diabetic tears compared to non-diabetic controls (P=0.006); IGF-1 levels were not significantly altered. No difference in corneal sensitivity was detected between groups. The concentration of IGFBP3 in tears was independent of IGF-1. Consistent with human tear measurements in vivo, IGFBP3 secretion was increased 2.2 fold (P<0.001) following culture of hTCEpi cells under elevated glucose conditions in vitro. Treatment with glucose and the mannitol control reduced IGFBP3 mRNA (P<0.001). Total IGF-1R levels were unchanged. The increase in the IGFBP3:IGF-1 ratio detected in diabetic tears compared to normal controls blocked phosphorylation of the IGF-1R by IGF-1 (P<0.001) when tested in vitro. Taken together, these in vivo and confirmatory in vitro findings suggest that the observed increase in IGFBP3 found in human tears may attenuate IGF-1R signaling in the diabetic cornea. A long-term increase in IGFBP3 may contribute to epithelial compromise and the pathogenesis of ocular surface complications reported in diabetes.

The impact of cellular debris on Pseudomonas aeruginosa adherence to silicone hydrogel contact lenses and contact lens storage cases.

OBJECTIVES: To evaluate neutrophil-enhanced Pseudomonas aeruginosa (PA) biofilm formation on silicone hydrogel contact lenses and to determine the effect of epithelial biodebris on PA adherence in contact lens storage cases. METHODS: A fully invasive PA corneal isolate stably conjugated to green fluorescent protein was used. Unworn lotrafilcon A contact lenses were incubated at various ratios of PA to polymorphonuclear neutrophil (PMN) for 24 hours at 37°C. Lens-associated PA was evaluated using laser scanning confocal microscopy and nonviable PA were visualized using propidium iodide. Viable bacteria were enumerated by colony-forming unit (CFU) analysis. For acute epithelial cell studies, PA viability was determined after coincubation with freeze-thaw epithelial cell lysates in 96-well polystyrene plates. Levels of residual cellular debris and bacterial viability were further assessed in used contact lens storage cases. RESULTS: Laser scanning confocal microscopy demonstrated that cotreatment with PMA-stimulated neutrophils increased PA adherence over 24 hours to lens surfaces with a striking alteration of PA architecture. Propidium iodide staining showed that the adherent bacteria consisted of a mixture of viable and nonviable PA; a PMN-associated increase in viable PA was confirmed by CFU (PA:PMN 0.1:1, P = 0.025; PA:PMN 1:1, P = 0.005). Acute epithelial cell debris studies revealed a significant increase in viable PA in 96-well plates in the presence of epithelial freeze-thaw lysates (PA:debris 1:1, P = 0.002; PA:debris 100:1, P = 0.002). Crystal violet staining of used lens storage cases revealed residual cellular debris at all time points, which was independent of microbial contamination; all lens cases used for periods of 9 months or more were uniformly associated with high levels of viable microorganisms. CONCLUSION: These results demonstrate that prolonged corneal inflammation with the presence of PMNs when confronted with simultaneous PA challenge in extended contact lens wear has the potential to stimulate biofilm formation on silicone hydrogel contact lenses. These findings further suggest that a persistent buildup of extracellular debris in lens storage cases may contribute to the heavy biofilms reported on these surfaces.

Hypoxia-induced downregulation of ΔNp63α in the corneal epithelium.

PURPOSE: The aim of this study was to establish a relationship between hypoxic stress and the expression of ΔNp63α in an established rabbit contact lens model and in cultured corneal epithelial cells. METHODS: New Zealand white rabbits were fit in one eye with either a non-oxygen transmissible or hyper-oxygen-permeable rigid contact lens for 24 hrs of wear; the contralateral eye was used as a control. All the rabbits underwent a bilateral nictitating membranectomy to facilitate lens retention. ΔNp63α expression was analyzed by immunofluorescence and western blot. Telomerase-immortalized human corneal epithelial cells (hTCEpi) were grown in serum-free media and treated with the hypoxia-mimetic cobalt chloride to simulate hypoxia for 6 hrs (short term) or 24 hrs (prolonged). Transcriptional activity and protein levels were assessed using luciferase reporter assays, reverse transcription polymerase chain reaction, and western blot. Cell viability was assessed by live/dead assay. RESULTS: Compared with the non-lens wearing eye, 24 hrs of non-oxygen transmissible lens wear in vivo decreased ΔNp63α protein levels in both the limbal and central corneal epithelium; this decrease was not found in the hyper-oxygen transmissible lens group. In hTCEpi cells in vitro, hypoxia increased the activity of the ΔN promoter but reduced the levels of ΔNp63α mRNA after 24 hrs of prolonged culture. Similarly, ΔNp63α expression levels were unaffected from short-term exposure but decreased after 24 hrs. Live/dead assay confirmed the presence of viable cells after CoCl2 treatment at 6- and 24-hr time points. Cells treated for 24 hrs were viable but were smaller and rounded with signs of membrane blebbing, consistent with early stages of apoptosis. CONCLUSIONS: Hypoxic stress induced by either prolonged wear of a nonoxygen transmissible lens in vivo or hypoxic-mimic conditions by cobalt chloride in vitro downregulates ΔNp63α in the corneal epithelium. The loss of ΔNp63α in response to hypoxic stress may contribute to the disruption of normal renewal mechanisms reported with low oxygen transmissible contact lens wear and prolonged eyelid closure.