UV light-blocking contact lenses protect against short-term UVB-induced limbal stem cell niche damage and inflammation

Abstract

UVB irradiation has been linked to pathogenesis of pterygium, a conjunctival tumor growing onto transparent cornea, the windscreen of the eye. Due to corneal anatomy, ambient UVB irradiation is amplified at the stem cell-containing nasal limbus. The aim of this study was to analyse the effect of a UV-blocking contact lens (UVBCL, senofilcon A, Class 1 UV blocker) on limbal epithelial cells and fibroblasts under UVB irradiation compared to a non-UVB-blocking contact lens. UVBCL prevented UVB-induced DNA damage (as assessed by cyclobutane pyrimidine dimer immunostaining) as well as a decrease in proliferation and scratch wound closure rate of both limbal epithelial and fibroblast cells. Similarly, UVBCL protected limbal epithelial cells from UVB-induced loss of their phenotype in terms of colony forming efficiency and stem cell marker expression (ABCB5, P63α, integrin β1) compared to controls. Moreover, with UVBCL pro-inflammatory cytokines such as TNFα and MCP1 remained unchanged. These data demonstrate the significance of UV-protection in preserving the limbal niche in response to at least short-term UVB. Our data support the use of UVBCL in protecting limbal niche cells, especially after limbal stem cell transplantation and in patients after pterygium surgery, to help prevent recurrences.

Figure 1

UVB blocking contact lenses effectively block UVA and UVB light. (A) Protective UV-absorbing eyewear, such as sunglasses, alone are not able to protect the ocular surface from UV rays coming from the side direction (depicted in yellow arrows) (B). UV blocking contact lenses offer total protection of the cornea and the limbus and when combined with UV protective eyeware the entire ocular surface is protected. The UV blocking contact lenses tested, ACUVUE OASYS (senofilcon A) offer 10-fold more protection for UVA and 17-fold for UVB, reaching 96% and 100% blocking of UVA (C) and UVB (D) respectively compared to contact lenses without a UV blocker, namely Air Optix Night & Day (lotrafilcon A). Graphics with courtesy from Johnson & Johnson Vision.

Figure 2

Senofilcon A UVB blocking contact lenses prevent UVB-induced formation of cyclobutane pyrimidine dimers (CPD, marker of DNA damage) in limbal epithelial cells. Representative immunofluorescence photos of CPD in limbal epithelial cells following UVB irradiation (20 mJ/cm²): BT (AbsorbMax black tape) (A) NO COVER (B), CL (C), UVBCL (D) and NO UV (E). (F) Quantified corrected total cell fluorescence (CTCF, n = 6, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

Senofilcon A UVB blocking contact lenses protect against UVB-induced cyclobutane pyrimidine dimer (CPD) formation in limbal fibroblasts. Representative immunofluorescence photos of CPD in limbal fibroblasts following UVB irradiation (20 mJ/cm²): BT (AbsorbMax black tape) (A) NO COVER (B), CL (C), UVBCL (D) and NO UV (E). (F) Quantified corrected total cell fluorescence (CTCF, n = 6, ***p < 0.001, ****p < 0.0001).

Figure 4

Senofilcon A UVB blocking contact lenses protect against changes in the metabolic activity and rate of scratch wound closure of limbal epithelial cells and fibroblasts. (A,B) Alamar Blue data showed that the metabolic activity of HLE (A) and HLF (B) cells which were protected by the UVBCL contact lens was maintained in similar levels to the BT and NO UV controls (20 mJ/cm²). (C,D) HLE cells exhibited non-significant changes in wound healing activity at neither 4 h nor 8 h time-point following UVB irradiation (C). HLF cells protected by CL contact lenses featured slower wound closure compared to cells covered by UVBCL lenses, BT and cells that were not UV treated (NO UV) (D). n = 6, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

UVB-induced reduction of limbal epithelial cell colony forming efficiency is prevented with protection by senofilcon A UVB blocking contact lenses. (A) Colony forming efficiency of irradiated limbal epithelial cells, either unprotected or covered with CL lenses, was significantly reduced compared to their non-irradiated counterparts indicating loss of proliferative potential as a result of ultraviolet B treatment. This is reversed by using UVBCL contact lenses to protect the HLE cells from UVB irradiation. Photos B-F depict representative photos of cultures, n = 6, ****p < 0.001.

Figure 6

UVB-induced changes of limbal epithelial cell phenotype is prevented with protection by senofilcon A UVB blocking contact lenses. Immunocytochemistry of limbal epithelial cell marker expression including p63α (Α,E,I,M,Q, Alexa488), ABCB5 (B,F,J,N,R, Alexa 555), β1 integrin (C,G,K,O,S, Alexa488) and keratin 3 (D,H,L,P,T). Limbal epithelial cells in the NO COVER and CL groups partially lost the expression of the markers integrin p63α (E,I), ABCB5, (F,J) β1integrin (G,K) while areas expressing the differentiation marker K3 increased (H,L, white arrows accentuated K3-positive regions). The immunocytochemistry data are quantified and summarized in graph (U,V,W,X), n ≥ 3, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bars correspond to 100 μm.

Figure 7

Senofilcon A UVB blocking contact lenses prevent modifications in the expression of key inflammation and angiogenesis-related proteins produced by limbal epithelial cells and limbal fibroblasts. ELISA analysis of conditioned media from limbal epithelial cells and limbal fibroblasts for TNFα (A), MCP-1 (B), VEGFA (C) and VEGFC (D). UVB irradiation induced an upregulation of TNFα and MCP-1 (in limbal epithelial cells and limbal fibroblasts respectively) while this was prevented by the use of UVBCL lenses to protect the cultures (A,B). UVB irradiation of limbal epithelial cells induced a reduction in VEGFA and VEGFC (C,D) while covering of the cultures with UVBCL lenses maintained the protein levels similar to the ones of the controls. (n = 3, *p < 0.05, **p < 0.01 and ***p < 0.001 and ****p < 0.0001). Four asterisks (****) situated above bars without brackets correspond to significance in comparison to all other groups. (● signifies that the protein levels were under the detectable levels of the assay).