PAX6, FABP5 and keratocytes‐characteristic marker expression in human limbal fibroblasts and keratocytes of congenital aniridia and healthy subjects, in vitro

Abstract

Aims/Purpose: To investigate PAX6, FABP5 and keratocyte‐characteristic marker expression in human limbal fibroblasts and keratocytes (LFCs and LKCs) of congenital aniridia (AN) and healthy corneas, in vitro.Methods: Primary human limbal fibroblasts (LFCs/ AN‐LFCs) were isolated from aniridia (n = 8) and healthy corneas (n = 8) and were cultured in DMEM/F12 with 5% fetal calf serum. Limbal keratocytes (LKCs/ AN‐LKCs) were cultured in low‐glucose serum‐free cell culture medium (LGSF‐medium). Stem cell marker PAX6 and ABCG2, retinoic acid marker FABP5 and keratocyte‐characteristic marker collagen I (COL1A1), collagen III (COL3A1), collagen V (COL5A1), lumican (LUM), keratocan (KER), α‐smooth muscle actin (ACTA2), CD34, aldehyde dehydrogenase 3 family, member A1 (ALDH3A1) expression analysis has been performed by qPCR and Western blot.Results: PAX6 mRNA expression was significantly lower in AN‐LFCs and AN‐LKCs than in LFCs and LKCs (p = 0.04; p = 0.014). FABP5 mRNA expression was significantly higher in LKC and AN‐LKCs than in LFCs and AN‐LFCs (p = 0.036; p = 0.012) COL5A1 mRNA expression was significantly lower (p = 0.007), KER and ALDH3A1 mRNA expression was significantly higher (p = 0.013; p = 0.007) in AN‐LFCs than in LFCs. COL1A1 and COL5A1 mRNA expression was significantly higher in AN‐LKCs than in LKCs (p = 0.009; p = 0.001). Protein expression did not differ significantly between LFCs/ AN‐LFCs or LKCs/ AN‐LKCs for any of the analysed markers (p ≥ 0.05).Conclusions: Our study confirmed altered gene expression in aniridia limbal fibroblasts and keratocytes compared to healthy controls. These may play an important role in development and progression of aniridia associated keratopathy.