Abstract
Purpose :
During corneal wound healing, corneal keratocytes are transformed into fibroblasts and/or myofibroblasts. Myofibroblasts produce collagen fibrils that are disorganized and result in corneal haze and scarring. Recently, vitamin D has been studied as an anti-fibrotic agent for hepatic fibrosis. We investigated the effects of 1,25- and 24,25-dihyroxyvitamin D3 (Vit D3) and vitamin D receptor knockout (VDR KO) on corneal stromal cell differentiation.
Methods :
Human and mouse corneal stromal cells were isolated and cultured in DMEM plus 1% FBS. Mouse cornea organ culture was also used. TGF β1 was used to stimulate myofibroblast differentiation. Stromal cells were cultured with 5 ng/ml TGF β1 for 72 h along with 20 nM 1,25- or 100 nM 24,25-Vit D3. Mouse corneas were cultured with 10 ng/ml TGF β1 for 72h. Total RNA and protein were isolated from corneas and from cultured primary corneal stromal cells. α-Smooth muscle actin (α-SMA) transcript levels were assessed by qPCR. Western blotting and immunochemistry were used to detect α-SMA protein levels.
Results :
1,25- and 24,25-Vit D3 significantly reduced α-SMA mRNA levels in whole mouse corneas cultured with TGF β1. 1,25-Vit D3 also reduced α-SMA protein expression in human and mouse primary corneal stromal cells cultured with TGF β1. 24,25-Vit D3 also significantly reduced α-SMA protein expression in human and mouse primary corneal stromal cells. α-SMA protein expression was also reduced in VDR KO mouse primary stromal cells cultured with 1,25- and 24,25-Vit D3. Similar results were detected with immunochemistry.
Conclusions :
Both 1,25- and 24,25-Vit D3 can inhibit TGF β1-inducing fibroblast-to-myofibroblast trans-differentiation. While 1,25-Vit D3 has been shown to inhibit fibrotic responses in tissues such as the liver, this is, to the best of our knowledge, the first time 24,25-Vit D3 has been shown to inhibit fibrosis. This is also the first demonstration of Vit D inhibiting fibrosis in VDR KO cells. The ability of both forms of Vit D to reduce myofibroblast trans-differentiation in cells derived from VDR KO mice indicate that the VDR is not essential for this response.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.