Abstract
Purpose :
Determine if endogenous TGFβ signaling is required to promote both normal wound healing and the fibrotic response to injury within an ex vivo mock cataract surgery model.
Methods :
For these studies an ex vivo mock cataract surgery chick wound healing/fibrosis model was used where the repair outcome to cataract surgery depends on the environment that the injury activated CD44+ leader cells encounter. On the fiber cell denuded lens basement membrane capsule, CD44+ cells lead the collective movement of the wounded lens epithelial cells to promote wound healing. CD44+ leader cells also direct the epithelial cells to move off the lens capsule into the extracellular zone (ECZ), a pro-fibrotic culture substrate. In the ECZ, CD44+ cells are induced to transition to fibrotic disease causing αSMA+ myofibroblasts by day 3 post-injury. To determine if endogenous TGFβ signaling is required for wound healing and myofibroblast differentiation, cultures were incubated +/- TGFβRI inhibitors. Wound healing was followed by phase contrast microscopy through its completion 3 days post-injury. Fibrotic markers and/or signaling molecules were analyzed by immunostaining or western blot analysis.
Results :
SMAD activation was observed 3hr post-injury, demonstrating that an endogenous TGFβ signaling pathway is activated in response to mock cataract surgery. The pharmacological inhibition of TGFβ signaling had no observable impact on collective migration of the lens epithelium across the cell-denuded basement membrane capsule and the wound was closed by day 3 post-injury. In this environment the CD44+ leader cells do not significantly alter their matrix microenvironment; no fibronectin EDA or collagen I was detected on the substrate surface. In contrast, when the CD44+ leader cells move across the ECZ, these cells produce a pro-fibrotic matrix that includes fibronectin EDA and collagen I on the substrate beneath the cells. These matrix elements are consistent with the transition of these cells to αSMA+ myofibroblasts. In this matrix environment, endogenous TGFβ produced in response to wounding is required for the fate change of these cells to αSMA+ myofibroblasts.
Conclusions :
The impact of the endogenous TGFβ response to cataract surgery injury is dependent upon the matrix microenvironment.
This is a 2020 ARVO Annual Meeting abstract.