Blue-light induced breakdown of barrier function on human retinal epithelial cells is mediated by PKC-ζ over-activation and oxidative stress

We aimed to study the time course decrease of human retinal pigment epithelium (RPE) barrier function when exposed to blue light. To this end, we cultured ARPE-19 cells on Electrical Cell-substrate Impedance Sensing (ECIS) multi-well arrays. Using an ad hoc light emitting diode (LED) array illumination system together with a set of neutral density filters and a 3-dimensional (3D) printed filter holder, cells were exposed to a gradient of irradiances of blue-light with a measured peak at 468 nm. The electrical resistance between 4 kHz and 64 kHz was recorded during the exposure. Blue light exposure induced a dose-dependent decrease in the resistances at 4 kHz, however the time course resistance at 64 kHz did not show any decrease before t = 52 h. Quantification of the barrier function using mathematical model integrated in the ECIS software showed that blue-light exposure induced a dose-dependent decrease in the barrier function associated with tight junction formation (P < 0.05). This was confirmed by the immunostaining of the tight-junction associated structural protein, Zonula occludens-1 (ZO-1). The detection of reactive oxygen species by carboxy-H2DCFDA confirmed that the blue light induced dose-dependent decrease in the barrier function is mediated by oxidative stress. On a separate experiment, blue-light exposed ARPE-19 cells were treated with 100 nM Protein Kinase C zeta (PKC-ζ) pseudo substrate inhibitor to identify underlying pathway for blue-light induced damage on the barrier function. The treatment with 100 nM PKC-ζ pseudo substrate inhibitor induced faster recovery of the barrier function compared to no treatment. Altogether our results document that blue LED light exposure decreased RPE barrier function in-vitro in a dose-dependent manner, before any cell death occurred. This damage induced by blue-light on tight junctions is mediated by oxidative stress through PKC-ζ activation. The quantification of the healing effect observed by inhibition of PKC-ζ might lead to development of high throughput wound healing assays through ECIS in the future.