Abstract
Epithelial wound healing is essential to repair the corneal barrier function after injury and requires coordinated epithelial sheet movement over the wounded region. The presence and role of pannexin1 on multilayered epithelial sheet migration was examined in unwounded and wounded corneal epithelium from C57BL/6J (B6) control and diet-induced obese (DiO) mice, a pretype 2 diabetic model. We hypothesize that pannexin1 is dysregulated, and the interaction of two ion-channel proteins (P2X7 and pannexin1) is altered in pretype 2 diabetic tissue. Pannexin1 was found to be present along cell borders in unwounded tissue, and no significant difference was observed between DiO and B6 control. However, an epithelial debridement induced a striking difference in pannexin1 localization. The B6 control epithelium displayed intense staining near the leading edge, which is the region where calcium mobilization was detected, whereas the staining in the DiO corneal epithelium was diffuse and lacked distinct gradation in intensity back from the leading edge. Cells distal to the wound in the DiO tissue were irregular in shape, and the morphology was similar to that of epithelium inhibited with 10Panx, a pannexin1 inhibitor. Pannexin1 inhibition reduced mobilization of calcium between cells near the leading edge, and MATLAB scripts revealed a reduction in cell-cell communication that was also detected in cultured cells. Proximity ligation was performed to determine if P2X7 and pannexin1 interaction was a necessary component of motility and communication. While there was no significant difference in the interaction in unwounded DiO and B6 control corneal epithelium, there was significantly less interaction in the wounded DiO corneas both near the wound and back from the edge. The results demonstrate that pannexin1 contributes to the healing response, and P2X7 and pannexin1 coordination may be a required component of cell-cell communication and an underlying reason for the lack of pathologic tissue migration.
Highlights
Obesity is a major risk factor for the development of type 2 diabetes
Anti-pannexin1 monoclonal mouse antibody directed against pannexin1 (Cat. #SC515941) was purchased from Santa Cruz Biotechnology (Dallas, TX), and anti-P2X7 polyclonal rabbit antibody targeting the extracellular domain of P2X7 receptors (Cat. #APR-008) was purchased from Alomone Labs. 10Panx, a pannexin1 mimetic inhibitory peptide (Cat. #3348), and its scrambled peptide were purchased from Tocris Biosciences (Minneapolis, MN)
These data led us to speculate that both P2X7 and pannexin1 may act as sensors at the leading edge of a wound; in tissue where wound healing is compromised, these sensors may not occur
Summary
Obesity is a major risk factor for the development of type 2 diabetes. The cornea is an excellent organ to examine the effects of diet on wound healing, as it is a relatively simple avascular tissue that is oxygenated via diffusion. Corneal epithelial repair requires a number of synchronized events that include, but are not limited to, cell motility, cell-cell communication, matrix deposition, and tissue remodeling [1, 2]. The apical corneal epithelium and its tight junctions become compromised, and large molecules and pathogens can penetrate into the epithelium. This impaired function appears to be exacerbated in patients with elevated HbA1c levels [3,4,5] and enhances the risk for delayed wound healing after injury, epithelial fragility, and possibly decreased corneal epithelial stiffness [6,7,8]. It was observed that the injury-induced release of nucleotides triggered a transient calcium (Ca2+)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
