Chronic Hyperglycemia Compromises Mitochondrial Function in Corneal Epithelial Cells: Implications for the Diabetic Cornea.

Mitochondrial dysfunction is a major pathophysiological event leading to the onset of diabetic complications. This study investigated the temporal effects of hyperglycemia on mitochondrial metabolism in corneal epithelial cells. To accomplish this, human telomerase-immortalized corneal epithelial cells were cultured in a defined growth medium containing 6 mM glucose. To simulate hyperglycemia, cells were cultured in a medium containing 25 mM D-glucose, and control cells were cultured in mannitol. Using metabolic flux analysis, there was a hyperosmolar-mediated increase in mitochondrial respiration after 24 h. By day 5, there was a decrease in spare respiratory capacity in cells subject to high glucose that remained suppressed throughout the 14-day period. Although respiration remained high through day 9, glycolysis was decreased. Mitochondrial respiration was decreased by day 14. This was accompanied by the restoration of glycolysis to normoglycemic levels. These changes paralleled a decrease in mitochondrial polarization and cell cycle arrest. Together, these data show that chronic but not acute hyperglycemic stress leads to mitochondrial dysfunction. Moreover, the hyperglycemia-induced loss of spare respiratory capacity reduces the ability of corneal epithelial cells to respond to subsequent stress. Compromised mitochondrial function represents a previously unexplored mechanism that likely contributes to corneal complications in diabetes.

Differential effects of obstructive sleep apnea on the corneal subbasal nerve plexus and retinal nerve fiber layer.

PURPOSE: Obstructive sleep apnea (OSA) is an established independent risk factor for peripheral neuropathy. Macro and microvascular changes have been documented in OSA, including high levels of potent vasoconstrictors. In diabetes, vasoconstriction has been identified as an underlying risk factor for corneal neuropathy. This study sought to establish a potential relationship between OSA and corneal nerve morphology and sensitivity, and to determine whether changes in corneal nerves may be reflective of OSA severity. DESIGN: Single center cross-sectional study. METHODS: Sixty-seven patients were stratified into two groups: those with OSA and healthy controls. Groups were matched for age, sex, race, smoking, and dry eye status. Outcome measures included serologies, a dilated fundus exam, dry eye testing, anthropometric parameters, corneal sensitivity, subbasal nerve plexus morphology, retinal nerve fiber layer (RNFL) thickness, and the use of questionnaires to assess symptoms of dry eye disease, risk of OSA, and continuous positive airway pressure (CPAP) compliance. RESULTS: No significant differences were observed in corneal nerve morphology, sensitivity, or the number of dendritic cells. In the OSA test group, RNFL thinning was noted in the superior and inferior regions of the optic disc and peripapillary region. A greater proportion of participants in the OSA group required a subsequent evaluation for glaucoma than in the control. In those with OSA, an increase in the apnea hypopnea index was associated with an increase in optic nerve cupping. CONCLUSIONS: OSA does not exert a robust effect on corneal nerves. OSA is however, associated with thinning of the RNFL. Participants with glaucomatous optic nerve changes and risk factors for OSA should be examined as uncontrolled OSA may exacerbate glaucoma progression.

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.

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.

IGFBP-3 Mediates Metabolic Homeostasis During Hyperosmolar Stress in the Corneal Epithelium.

Purpose: The insulin-like growth factor binding protein-3 (IGFBP-3) is a multifunctional secretory protein with well-known roles in cell growth and survival. Data in our laboratory suggest that IGFBP-3 may be functioning as a stress response protein in the corneal epithelium. The purpose of this study is to determine the role of IGFBP-3 in mediating the corneal epithelial cell stress response to hyperosmolarity, a well-known pathophysiological event in the development of dry eye disease. Methods: Telomerase-immortalized human corneal epithelial (hTCEpi) cells were used in this study. Cells were cultured in serum-free media with (growth) or without (basal) supplements. Hyperosmolarity was achieved by increasing salt concentrations to 450 and 500 mOsM. Metabolic and mitochondrial changes were assessed using Seahorse metabolic flux analysis and assays for mitochondrial calcium, polarization and mtDNA. Levels of IGFBP-3 and inflammatory mediators were quantified using ELISA. Cytotoxicity was evaluated using a lactate dehydrogenase assay. In select experiments, cells were cotreated with 500 ng/mL recombinant human (rh)IGFBP-3. Results: Hyperosmolar stress altered metabolic activity, shifting cells towards a respiratory phenotype. Hyperosmolar stress further altered mitochondrial calcium levels, depolarized mitochondria, decreased levels of ATP, mtDNA, and expression of IGFBP-3. In contrast, hyperosmolar stress increased production of the proinflammatory cytokines IL-6 and IL-8. Supplementation with rhIGFBP-3 abrogated metabolic and mitochondrial changes with only marginal effects on IL-8. Conclusions: These findings indicate that IGFBP-3 is a critical protein involved in hyperosmolar stress responses in the corneal epithelium. These data further support a new role for IGFBP-3 in the control of cellular metabolism.

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.

The IGF/Insulin-IGFBP Axis in Corneal Development, Wound Healing, and Disease.

The insulin-like growth factor (IGF) family plays key roles in growth and development. In the cornea, IGF family members have been implicated in proliferation, differentiation, and migration, critical events that maintain a smooth refracting surface that is essential for vision. The IGF family is composed of multiple ligands, receptors, and ligand binding proteins. Expression of IGF type 1 receptor (IGF-1R), IGF type 2 receptor (IGF-2R), and insulin receptor (INSR) in the cornea has been well characterized, including the presence of the IGF-1R and INSR hybrid (Hybrid-R) in the corneal epithelium. Recent data also indicates that each of these receptors display unique intracellular localization. Thus, in addition to canonical ligand binding at the plasma membrane and the initiation of downstream signaling cascades, IGF-1R, INSR, and Hybrid-R also function to regulate mitochondrial stability and nuclear gene expression. IGF-1 and IGF-2, two of three principal ligands, are polypeptide growth factors that function in all cellular layers of the cornea. Unlike IGF-1 and IGF-2, the hormone insulin plays a unique role in the cornea, different from many other tissues in the body. In the corneal epithelium, insulin is not required for glucose uptake, due to constitutive activation of the glucose transporter, GLUT1. However, insulin is needed for the regulation of metabolism, circadian rhythm, autophagy, proliferation, and migration after wounding. There is conflicting evidence regarding expression of the six IGF-binding proteins (IGFBPs), which function primarily to sequester IGF ligands. Within the cornea, IGFBP-2 and IGFBP-3 have identified roles in tissue homeostasis. While IGFBP-3 regulates growth control and intracellular receptor localization in the corneal epithelium, both IGFBP-2 and IGFBP-3 function in corneal fibroblast differentiation and myofibroblast proliferation, key events in stromal wound healing. IGFBP-2 has also been linked to cellular overgrowth in pterygium. There is a clear role for IGF family members in regulating tissue homeostasis in the cornea. This review summarizes what is known regarding the function of IGF and related proteins in corneal development, during wound healing, and in the pathophysiology of disease. Finally, we highlight key areas of research that are in need of future study.

Development and Implementation of Psychiatric Services in a Student-Operated Clinic.

This article chronicles the development and implementation of Monday Psychiatry Clinic, a psychiatry component of a medical student-operated free clinic (The Monday Clinic) in Dallas, Texas, providing assessment data systematically collected in the clinic. The established clinical purpose of Monday Psychiatry Clinic is to assess common psychiatric disorders and refer patients in need to appropriate sources of care. This clinic provided leadership education and volunteering opportunities to medical students who learned to interact with patients with psychiatric concerns, established clinical interviewing skills, and became familiar with psychiatric diagnostic criteria. More than one-third of the patients screened positive for depression, alcohol, or drug problems, and one-fourth of the patients with a positive screen were diagnosed with one of these disorders. All patients with identified problems were referred for the appropriate level of care.

Tear Levels of Insulin-Like Growth Factor Binding Protein 3 Correlate With Subbasal Nerve Plexus Changes in Patients With Type 2 Diabetes Mellitus.

Purpose: This study investigated the expression of insulin-like growth factor binding protein-3 (IGFBP-3) in basal tears of patients with type 2 diabetes mellitus compared to nondiabetic controls; and correlated tear levels of IGFBP-3 with morphologic changes in the subbasal nerve plexus and density of basal corneal epithelial cells. Methods: This was a single visit, cross-sectional study. Diabetic and control subjects were matched for age, sex, smoking status, and body mass index. Tear levels of IGFBP-3 were measured using ELISA. Corneal nerve and basal epithelial cell changes were measured using in vivo confocal microscopy. Results: Tear levels of IGFBP-3 were 3.5-fold higher in those with diabetes. Patients with diabetes also showed a reduction in nerve fiber layer, nerve branch density, and corneal basal epithelial cell density. There was no significant difference in corneal sensitivity. IGFBP-3 levels were highly correlated with nerve fiber length and branch density; while hemoglobin (Hb)A1c was only moderately correlated. There were no significant differences in the clinical or subjective signs of dry eye between groups, indicating that tear levels of IGFBP-3 and corneal nerve changes were not due to the presence of mild dry eye. Conclusions: These findings indicate that tear levels of IGFBP-3 are more tightly correlated to nerve fiber changes in diabetes than HbA1c. Future studies that stratify the severity of diabetic disease with tear levels of IGFBP-3 are needed to validate this finding.

Alterations in corneal nerves following crack cocaine use mimic diabetes-induced nerve damage.

SUMMARY: The use of in vivo confocal microscopy (IVCM) is rapidly emerging as an important clinical tool to evaluate changes in corneal sensory nerves as a surrogate measure for diabetic peripheral neuropathy. Commonly used metrics to document and grade the severity of diabetes and risk for diabetic peripheral neuropathy include nerve fiber length, density, branching and tortuosity. In addition to corneal nerves, thinning of the retinal fiber layer has been shown to correlate with the severity of diabetic disease. Here, we present a case report on a pre-diabetic 60-year-old native American woman with abnormal corneal nerve morphology and retinal nerve fiber layer thinning. Her past medical history was positive for illicit substance abuse. IVCM showed a decrease in nerve fiber density and length, in addition to abnormally high levels of tortuosity. OCT revealed focal areas of reduced retinal nerve fiber layer thickness that were asymmetric between eyes. This is the first report of abnormally high levels of tortuosity in the corneal sub-basal nerve plexus in a patient with a past history of cocaine abuse. It also demonstrates, for the first time, that illicit substance abuse can have long-term adverse effects on ocular nerves for years following discontinued use of the drug. Studies using IVCM to evaluate changes in corneal nerve morphology in patients with diabetes need to consider a past history of illicit drug use as an exclusionary measure. LEARNING POINTS: Multiple ocular and systemic factors can impede accurate assessment of the corneal sub-basal nerve plexus by IVCM in diabetes.Although current history was negative for illicit substance abuse, past history can have longstanding effects on corneal nerves and the retinal nerve fiber layer.Illicit drug use must be considered an exclusionary measure when evaluating diabetes-induced changes in corneal nerve morphology and the retinal nerve fiber layer.