Expression and Role of Insulin-like Growth Factor 2 Receptor (IGF2R) in the Cornea in Response to Wound Healing

Abstract

 

Purpose

 

IGF2R associates with ligands that influence wound healing outcomes such as the TGFβ Receptor, IGF-2, and CTGF, however, the expression pattern of this receptor in the cornea is unknown. We hypothesize that IGF2R regulates corneal stromal fibroblast to myofibroblast differentiation.

 

Methods

 

Human keratocytes were isolated from donor corneas. Fibroblasts (FGF-2 treated) or myofibroblasts (TGFβ-treated) were analyzed for IGF2R expression by Western blot. Mouse corneas were wounded in vivo and porcine corneas ex-vivo. Immunohistochemistry was used to quantify IGF2R and α-SMA protein expression after wounding compared to control. Lentivirus shRNAi was used to knock down the expression of IGF2R in human corneal fibroblasts and the extent of TGF-β1-stimulated differentiation to myofibroblasts was determined by α-smooth muscle actin (α-SMA) immunolocalization.

 

Results

 

Immunohistochemistry of corneal tissue demonstrate that IGF2R is expressed in epithelial, stromal and endothelial cells of normal human, mouse and porcine corneas. Double-staining with α-SMA and IGF2R-specific antibodies revealed the dramatic increase in IGF2R expression observed in the stroma of the wounded cornea occurs in myofibroblasts (see below). Western blot analyses of human primary stromal keratocytes incubated with FGF-2 or TGF-β1 in vitro demonstrate increased expression (2.9 ± 0.7-fold) of IGF2R in myofibroblasts relative to fibroblasts. Conversion of IGF2R shRNA-lentiviral particle transduced corneal fibroblasts to myofibroblasts reveals a dependence on IGF2R expression, as only 20 - 60% of cells transduced with shRNAi-lentiviral particles converted to myofibroblasts compared to 80% or greater in control cells in three separate trials. The table below summarizes the results for corneal stromal cells from three patients.

 

Conclusions

 

IGF2R protein expression is increased during corneal wound healing and regulates fibroblast to myofibroblast differentiation.

 

 

Untreated porcine corneas or those wounded with a 5mm trephine into the stroma were mounted on a base of 1% agarose and 1mg/ml collagen and placed in organ culture. After 2 weeks, the corneas were fixed, and sections were double-labeled with antibodies to IGF2R and αSMA. Nuclei were stained with DAPI. Bars, 100µm

 

 

The percentages shown are the average for the indicated number (n) of individual micrographs with 150 - 200 cells per micrograph were counted.

 

Keywords: 484 cornea: stroma and keratocytes • 480 cornea: basic science • 482 cornea: epithelium