Impact of membrane associated mucin and diquafosol on the wettability of human corneal epithelium cell layers

Abstract

Purpose: The integrity of corneal epithelium glycocalyx is considered of key importance for the wetting of the ocular surface and for the tear film stability. In order to quantitatively evaluate the contribution of membrane associated mucin, MAM, to corneal wetting the changes in the wettability were measured of human corneal epithelium cell (HCEC) layers: (i) intact, (ii) after shedding of MAM (the glycocalyx main constituent) and (iii) subsequent treatment with diquafosol, DQS (MAM expression enhancing drug).

Methods: HCEC cells were cultured to a confluent layer in Keratinocyte-SFM media subsequently switched to DMEM-F12 with 10% FBS and 10 ng/ml EGF. MAM shedding was performed via 30 min exposition to 5 µg/ml neutrophil elastase (NE). The NE-exposed cells were then treated for 1 h with 3% DQS. HCEC viability at each step was verified by MTT test. MAM expression was evaluated via immunofluorescence (OC125 targeted Anti-MUC16 conjugated with Alexa Fluor® 488 Goat Anti-Mouse IgG) and rose bengal (RB) staining. The wettability of HCEC layers kept in solution was probed through measuring of the equilibrium (θ_E), advancing (θ_A) and receding (θ_R) contact angles (CA) by axisymmetric sessile bubble shape analysis. At least 7 measurements were performed with each sample, 1 h and 24 h after treatments.

Results: Intact HCEC cells displayed continuous glycocalyx, resistance to RB staining and low CA (θ_E=34.03°±3.61° (SD), θ_A=37.08°±3.12°, θ_R=32.17°±2.99°). NE-exposed HCEC layers revealed decreased MAM expression, increased RB staining and significant (P<0.05) CA increase (θ_E=43.60°±3.15°, θ_A=48.87°±3.17°, θ_R=40.346°±3.08° at 1 h after treatment; θ_E=39.78°±2.48°, θ_A=41.097°±2.34°, θ_R=36.27°±2.55° at 24 h after treatment). The DQS treatment of NE-exposed cells resulted in prompt recovery of HCEC layers’ MAM expression, resistance to RB staining and CA (θ_E=36.87°±2.57°, θ_A=40.23°±2.47°, θ_R=35.16°±2.07° at 1 h after treatment and full recovery after 24 h).

Conclusions: The impact of MAM to the normal wettability of corneal epithelium surface is quantitatively evaluated. The wettability replenishment effect of DQS correlates with the data on its capability to rapidly enhance mucin expression in vivo. The relationship of corneal epithelium wettability with MAM integrity might be a major mechanism involved in tear film instability and in dry eye syndrome.