Deposition of matrix proteins during development and repair is critical to the transparency of the cornea. While many cells respond to a hypoxic state that can occur in a tumor, the cornea is exposed to hypoxia during development prior to eyelid opening and during the diurnal sleep cycle where oxygen levels can drop from 21% to 8%. In this study, we used 2 three-dimensional (3-D) models to examine how stromal cells respond to periods of acute hypoxic states. The first model, a stromal construct model, is a 3-D stroma-like construct that consists of human corneal fibroblasts (HCFs) stimulated by a stable form of ascorbate for 1, 2, and 4 weeks to self-assemble their own extracellular matrix. The second model, a corneal organ culture model, is a corneal wound-healing model, which consists of wounded adult rat corneas that were removed and placed in culture to heal. Both models were exposed to either normoxic or hypoxic conditions for varying time periods, and the expression and/or localization of matrix proteins was assessed. No significant changes were detected in Type V collagen, which is associated with Type I collagen fibrils; however, significant changes were detected in the expression of both the small leucine-rich repeating proteoglycans and the larger heparan sulfate proteoglycan, perlecan. Also, hypoxia decreased both the number of Cuprolinic blue-positive glycosaminoglycan chains along collagen fibrils and Sulfatase 1, which modulates the effect of heparan sulfate by removing the 6-O-sulfate groups. In the stromal construct model, alterations were seen in fibronectin, similar to those that occur in development and after injury. These changes in fibronectin after injury were accompanied by changes in proteoglycans. Together these findings indicate that acute hypoxic changes alter the physiology of the cornea, and these models will allow us to manipulate the conditions in the extracellular environment in order to study corneal development and trauma.

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of studies, the effects that environmental changes have on this form of movement are poorly understood. To examine the role of substrate compliancy on multi-layered epithelial sheet migration, we performed traction force and confocal microscopy to determine differences in traction forces and to examine focal adhesions on synthetic and biological substrates. The leading edges of corneal epithelial sheets undergo retraction or contraction prior to migration, and alterations in the sheet's stiffness are affected by the amount of force exerted by cells at the leading edge. On substrates of 30 kPa, cells exhibited greater and more rapid movement than on substrates of 8 kPa, which are similar to that of the corneal basement membrane. Vinculin and its phosphorylated residue Y1065 were prominent along the basal surface of migrating cells, while Y822 was prominent between neighboring cells along the leading edge. Vinculin localization was diffuse on a substrate where the basement membrane was removed. Furthermore, when cells were cultured on fibronectin-coated acrylamide substrates of 8 and 50 kPa and then wounded, there was an injury-induced phosphorylation of Y1065 and substrate dependent changes in the number and size of vinculin containing focal adhesions. These results demonstrate that changes in substrate stiffness affected traction forces and vinculin dynamics, which potentially could contribute to the delayed healing response associated with certain corneal pathologies.

The cornea is an excellent model tissue to study how cells adapt to periods of hypoxia as it is naturally exposed to diurnal fluxes in oxygen. It is avascular, transparent, and highly innervated. In certain pathologies, such as diabetes, limbal stem cell deficiency, or trauma, the cornea may be exposed to hypoxia for variable lengths of time. Due to its avascularity, the cornea requires atmospheric oxygen, and a reduction in oxygen availability can impair its physiology and function. We hypothesize that hypoxia alters membrane stiffness and the deposition of matrix proteins, leading to changes in cell migration, focal adhesion formation, and wound repair. Two systems-a 3D corneal organ culture model and polyacrylamide substrates of varying stiffness-were used to examine the response of corneal epithelium to normoxic and hypoxic environments. Exposure to hypoxia alters the deposition of the matrix proteins such as laminin and Type IV collagen. In addition, previous studies had shown a change in fibronectin after injury. Studies performed on matrix-coated acrylamide substrates ranging from 0.2 to 50 kPa revealed stiffness-dependent changes in cell morphology. The localization, number, and length of paxillin pY118-and vinculin pY1065-containing focal adhesions were different in wounded corneas and in human corneal epithelial cells incubated in hypoxic environments. Overall, these results demonstrate that low-oxygenated environments modify the composition of the extracellular matrix, basal lamina stiffness, and focal adhesion dynamics, leading to alterations in the function of the cornea.

Corneal disease and injury remain a major cause of blindness according to the World Health Organization. Certain conditions such as diabetes, corneal infection, and corneal ulceration may cause the cornea to be exposed to a hypoxic state for extended periods of time. Over 95% of the new cases of diabetes are Type II. In these patients, a simple wound can lead to complications which are compounded by the reduced ability to recover from hypoxic stress. An ex vivo corneal organ culture model was employed to examine cell migration. After a 24 hour exposure to hypoxia healing is delayed by 6 hours compared to control. In addition, there is a morphological change at the leading edge of cells. We hypothesize that hypoxia affects cell migration and wound healing by altering the regulation of basal lamina proteins and the turnover of focal adhesions. We determined that wounded rat corneas exposed to a hypoxic environment display decreased epithelial expression of syndecan‐1 and fibronectin. The localization of fibronectin throughout the epithelium and stroma is altered in the hypoxic cornea. Additional studies examined the alterations in expression and co‐localization of focal adhesion proteins. Hypoxia caused a change in the localization of vinculin and paxillin along the basal lamina and within the epithelium. Furthermore, there was a change in localization along the basal lamina of Y118 paxillin in the hypoxic cornea. By determining the changes in basal lamina composition and characterizing the changes in regulation and dynamics of focal adhesion proteins, we aim to improve the treatment of delayed wound healing associated with corneal hypoxia.