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Masaaki Ishii, Baerbel Rohrer; Partial Bystander Effect Elicited by single cell photo-oxidative blue light stimulation and apoptotic cell death radiation mediated by ROS and Calcium Signaling.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2708. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The “bystander effect” in biology refers to the phenomenon of induction of biological effects in cells not directly targeted, and may be mediated by gap junction (GJ) . Age-related macular degeneration (AMD) is associated with formation of drusen under the macular retinal pigment epithelium (RPE) cells. Interestingly, drusen, followed by geographic atrophy occurs focally in many patches which grow and subsequently coalesce. However, the cellular signaling pathway(s) involved in this non-uniform spreading of information and the mechanism(s) underlying the sparing of certain locations is unknown. Here we investigated spreading of reactive oxygen species (ROS) and calcium signals in RPE cell networks.
ARPE-19 cells were cultured for 14 days on glass-bottom culture dishes to establish well-connected monolayers. CellRox Green and H2DCFDA were used to detect ROS, Fluo8 AM to indicate calcium, tetramethylrhodamine to indicate mitochondrial membrane potential (ψm) and TO-Pro3 to indicate dead cells. GJs were blocked using 1-octanol. For data acquisition and photo-stimulation, the UltraView Vox live imaging system (Volocity software) was used; data was analyzed using Igor Pro.
Oxidative stress was initiated in individual cells using photo-stimulation with a 488 nm laser spot at 1 Hz. This stimulus led to changes in ROS, calcium and ψm in the affected cell. The calcium signaling was transmitted to neighboring cells slowly and in a non-uniform way; whereas the ROS signal spread fast and radially. Increased calcium levels were associated with a loss in ψm. GJ blockers prevented the spreading of the calcium but not the ROS-related signal. Importantly, the GJ-mediated calcium wave was associated with the induction of cell death by 24 hours. Cell death was correlated with baseline calcium levels.
These results demonstrate that local oxidative stress in a donor cell can trigger changes in selective connected recipient cells. It is unclear what leads to this patchiness, however, it suggests that damage occurs in susceptible areas, possibly associated with baseline calcium homeostasis, and is delayed in more resilient areas.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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