Abstract
Purpose :
To determine the role of FGFR2-mediated signaling in keratocytes during corneal development by phenotypic characterization of stromal specific FGFR2 knockout (KO) mouse
Methods :
In vitro analysis of FGFR2 knockdown was performed in cultured normal corneal keratocytes using Silencer® Select FGFR2 siRNA and siCtr (control), based on manufacture’s protocol. The cells were processed after 48 h post-transfection. The immunohistochemical analysis of FGFR2 knockdown in cells was determined by confocal microscopy. To determine the in vivo effect of FGFR2, a FGFR2 KO mouse model was generated. FGFR2 flox mice (Jackson Labs) were crossed with inducible keratocyte specific-Cre mice (Kera-rtTA/tet-O-Cre, Dr. Winston Kao, University of Cincinnati). The pregnant females were fed doxycycline chow (600 mg/kg) to induce tissue-specific FGFR2 KO pups. The flox-, Cre-, and wild-type (wt) mice were used as controls. Corneal thickness were determined using an ultrahigh resolution spectral domain optical coherence tomography instrument (SDOCT, Bioptigen).
Results :
In vitro, FGFR2 gene silencing severely inhibited pluripotency of stromal keratocytes when cultured in the presence of FGF2 (FGFR2-ligand) compared to control siRNA. The FGFR2 gene silenced-cells also exhibited pluripotency in the presence of IGF, suggesting that FGFR2 was needed for the pluripotency maintenance of keratocytes. OCT-based analysis of the FGFR2 KO mice corneas showed relatively thinner cornea compared to control mice. Immunohistochemical analysis also showed thinner cornea in FGFR2 mice compared to the control mice. Collagen-1 immunostaining was significantly down-regulated in KO mice corneal stroma compared to control mice.
Conclusions :
Our earlier results showed that FGFR2 was significantly downregulated in keratoconus corneal stroma. In vitro analysis showed FGFR2 knockdown affected the pluripotency of stromal keratocytes. The FGFR2 KO mice showed stromal thinning. Suggesting that the stromal-specific conditional FGFR2 KO mouse model will provide the functional understanding of FGFR2 in stromal keratocytes and also serve as a keratoconus animal model.
This is a 2021 ARVO Annual Meeting abstract.