Effect of the ROCK inhibitor Y-27632 on Proliferation and Differentiation of Equine Bronchial Epithelial Cells

Abstract

The airway epithelium plays an important role in physiological and pathological processes including inflammation, innate immunity and regenerative responses in the respiratory tract with regard to airway diseases such as human asthma, COPD and Equine Recurrent Airway Obstruction (RAO). Airway epithelial cell lines which would serve as an in vitro cell model are, indeed, missing for the horse. The objective of the study is to establish long-term equine bronchial epithelial cell cultures using the ROCK inhibitor Y-27632 and characterize them. Fresh isolated EBEC were cultured in the presence and absence of 10µM Y-27632 under conventional and air-liquid-interface culture conditions. Cell viability, morphology, proliferation and differentiation states were examined. Under conventional culture, Y-27632 induced higher growth rate of primary EBEC over a short period of time when compared to non-treated cells and increased the passage number. Epithelial cells grew faster and retain their epithelial cell morphology for a number of passages, with less contamination with fibroblasts. EBEC cultured on membrane inserts revealed high TEER values in the presence of Y-27632 and switching to Air-Liquid-Interface (ALI) did not cause decrement of TEER values. Inmunostaing of EBEC showed a different pattern of CK, TJP-1 and Vim expression indicating rapid differentiation of Y-27632-treated cells. Also, light and scanning electron microscopic imaging showed high amount of cilia, microvilli and ridges as well as more defined polygonal-shaped epithelial cells in the presence of Y-27632. Taken together, these results suggest that the ROCK inhibitor Y-27632 permits establishing conditional long-term in vitro airway epithelial cell models to investigate their role in equine airway diseases and as pharmacological targets.