The role of connective tissue growth factor, transforming growth factor and Smad signaling pathway during corneal wound healing

Abstract

To study the expression and location of connective tissue growth factor (CTGF) and transforming growth factor-beta(1) (TGF-beta(1)) protein and mRNA in rabbit cornea during the wound healing process. To assess the interaction between CTGF and TGF-beta(1), as well as the Smad signaling pathway involved. Twenty-six Albino white rabbits were used as experimental animals and randomly divided into 4 groups: (1) CONTROL GROUP: two rabbits. (2) Simple corneal injury group: a 3 mm diameter and 0.05 mm depth corneal tissue was excised by a trephine at the anterior central cornea as a corneal wound model in 12 rabbits. Two rabbits were randomly sacrificed at 2 h, 6 h, 1 d, 3 d, 7 d and 21 d after the trauma. (3) TGF-beta(1) antibodies treated group: 6 rabbits were injected with TGF-beta(1) antibodies (15.5 microg) subconjunctivally after corneal trephine. Two rabbits were randomly sacrificed at 3 d, 7 d and 21 d after the injection. (4) Smad4 antibodies treated group: 6 rabbits were injected with Smad4 antibodies (20 microg) subconjunctivally after corneal trephine. Two rabbits were randomly sacrificed at 3 d, 7 d and 21 d after the injection. Protein of CTGF, TGF-beta(1), and FN was assessed with immunohistochemistry. CTGF and type one collagen mRNA were measured in by in situ hybridization. (1) CTGF protein or mRNA did not exist in normal rabbit corneas, but TGF-beta(1) protein was expressed in normal rabbit cornea epithelium. (2) Cornea fibroblasts activated 6 h after the operation. Expression of CTGF, TGF-beta(1), FN protein and mRNA of CTGF and type one collagen were upregulated after cornea injury, and reached the highest level in 3 days. The expression was reduced to the basal level 21 days later. (3) Injection of TGF-beta(1) antibodies reduced the expression of CTGF, TGF-beta(1) and FN in the cornea stroma and down-regulated the expression of CTGF in corneal epithelial cells. (4) Injection of Smad4 antibodies inhibited the expression of TGF in the stroma but did not change the expression of CTGF. Injury can upregulate the expression of CTGF, TGF-beta(1), FN and type one collagen proteins and mRNA of CTGF and TGF-beta(1). TGF-beta(1) and CTGF affect the wound healing process. TGF can induce the activation of corneal fibroblasts and upregulate the expression of CTGF. CTGF modulates the production of FN and Type one collagen. These data support the hypothesis that TGF-beta(1) and CTGF promote cornea scar formation and imply that the regulation of CTGF and TGF-beta(1) synthesis may be a therapy target for reducing corneal scarring.