Smad4 deficiency ameliorates the progressive corneal stroma thinning caused by the loss of Tbr1

To understand how Tbr1 and Smad4 play a pivotal role in controlling ECM synthesis versus degradation for maintaining corneal stromal homeostasis and otherwise leading to corneal ectasia.

Keratocyte-specific and inducible knockout (iKO) of Tbr1, Smad4, or Tbr1/Smad4 double KO (iDKO) mice were generated. OCT was used to assess corneal thickness in vivo. Masson's trichrome and collagen hybridizing peptide stainings were performed to examine collagen expression. Immunostaining with an anti-cathepsin B antibody was used to assess ECM degradation. Cathepsin B inhibitor, CA-074Me, eyedrop was conducted to test its effect on treating stromal thinning in Tbr1 iKO mice.

Tbr1 iKO and Smad4 iKO displayed corneal thinning, but Tbr1 iKO revealed a progressive and more severe pathology than Smad4 iKO. Tbr1 iKO cornea lost most of its stroma and thus a dome shape. Collagen ECM is evenly distributed in Smad4 iKO as well as control littermates but was lost mainly in the anterior stroma of the Tbr1 iKO. Interestingly, Tbr1/Smad4 iDKO ameliorated Tbr1 iKO phenotype. The basal level of Cathepsin b (Ctsb) could be detected in the control stroma but was significantly increased in the Tbr1 iKO stromal cells and this effect was canceled in Tbr1/Smad4 iDKO. CA-074Me eyedrops administration significantly inhibited progressive corneal thinning caused by the Tbr1 iKO.

Our data from Tbr1/Smad4 iDKO argued that Smad4 played a pivotal role in controlling Tbr1-dependent ECM synthesis and Tbr1-independent ECM degradation to maintain corneal stromal integrity and homeostasis.