Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells' native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Activation of SRC kinases signals induction of posterior capsule opacification.

PURPOSE: Posterior capsule opacification (PCO) is a complication of cataract surgery resulting from the proliferation, migration, and epithelial-to-mesenchymal transition (EMT) of lens epithelial cells that remain associated with the lens capsule. These changes cause a loss of vision. The authors developed a chick embryo lens capsular bag model to study mechanisms involved in the onset of PCO. Because Src family kinases (SFKs) signal cell proliferation, migration, and EMT, the authors examined whether the inhibition of SFKs can prevent PCO. METHODS: After mock cataract surgery, chick lens capsular bags were pinned to a culture dish and grown in the presence or absence of the SFK inhibitor PP1. Cell movement was followed by photomicroscopy. Progression of proliferation and EMT in the PCO cultures was determined by Western blot analysis and immunofluorescence staining. RESULTS: As occurs in PCO, lens cells in this model proliferated, migrated across the posterior capsule, and expressed EMT markers, alpha-smooth muscle actin (alpha-SMA), and fibronectin (FN). Lens cells treated with PP1 maintained an epithelial phenotype, accumulated cadherin junctions, and did not migrate to the posterior capsule, increase proliferation, or express EMT markers. Therefore, exposure to PP1 prevented PCO. Short-term inhibition of SFKs was sufficient to prevent EMT, but longer inhibition was necessary to prevent lens cell migration. CONCLUSIONS: Progression of PCO involved early activation of SFKs. Lens cell migration preceded EMT, and each of these two events required activation of an SFK signaling pathway. Suppression of SFK activation blocked PCO, suggesting SFKs as a therapeutic target for the prevention of PCO.