Hypoxia Alters The Ca2+ Mobilization Of The Corneal Epithelium Upon Injury

Abstract

Purpose: : Previously, we demonstrated that the Ca²⁺ mobilization promotes corneal epithelium wound healing, such as cell migration. This process is thought to be modulated by the purinergic receptor pathway. Currently, the effect of hypoxia on the Ca²⁺ mobilization is largely unknown. We used human corneal limbal epithelial cells (HCLEs) and an organ culture to test our hypothesis that hypoxia is an early event in pathology, which leads to poor corneal wound healing. Our goal was to delineate changes in the mobilization of Ca²⁺ caused by hypoxia.

Methods: : HCLEs and corneal organ cultures were incubated under hypoxic conditions. Cells were pre-loaded with 10 μM acetoxymethyl (AM) ester derivatives of fluorescent indicators, and Ca²⁺ mobilization was induced by mechanical stimulation. Similar experiments were performed either in an extracellular Ca²⁺-free environment or in the presence of an intracellular Ca²⁺ chelator, BAPTA-AM. Functionality of gap junctions was evaluated under hypoxia using Fluorescence Recovery after Photobleaching (FRAP) and localization of Cx43 analyzed. P2X7 mRNA was examined by Real-Time PCR using TaqMan probes. Data analysis used the ΔΔCt method and values were given as mean +/- SEM using Student’s t-test (P<0.05). A circular abrasion was made on central cornea and organ cultures were incubated under hypoxic conditions. They were frozen-sectioned and F-actin filaments were detected using rhodamine phalloidin. All samples were performed in triplicate.

Results: : A decrease in oxygen did not change the maximum recovery of the Ca²⁺ response. However there was a significant decrease in the distance that the Ca²⁺ wave traveled (P<0.05, t-test). In the absence of external Ca²⁺, hypoxic conditions completely abrogated Ca²⁺ mobilization. In addition, incubation with BAPTA-AM resulted in attenuated Ca²⁺ propagation as seen in hypoxia. Localization of Cx43 did not change and FRAP demonstrated that hypoxia impaired the gating of gap junctions. P2X7 mRNA increased with hypoxia, while P2Y2 mRNA did not change. Cell migration was delayed in organ cultures under hypoxic conditions.

Conclusions: : Our results demonstrate that hypoxia alters Ca²⁺ propagation, a critical step for corneal wound healing. This is supported by the reduction in the functionality of gap junctions. Furthermore, the upregulation of P2X7 mRNA indicates that hypoxia alters the P2X7 signaling pathway, which is directly correlated with the recruitment of Ca²⁺ from the external source. Together, these suggest that alterations in Ca²⁺ response may lead to potential changes in downstream pathways involved in cell migration, and cause delayed corneal epithelium wound healing.

Keywords: cornea: epithelium • hypoxia • calcium