Abstract
Purpose :
Proliferative vitreoretinopathy (PVR) is the growth and contraction of cellular membranes within the vitreous cavity and on both surfaces of the retina and is the most common cause for recurrent retinal detachments. Cigarette smoke is associated with higher rates of PVR and our lab previously demonstrated elevations in proinflammatory cytokines TNF-α, IL-6, and IL-8 when RPE cells were exposed to cigarette smoke. Prior studies have shown that TNF-α induces RPE cells to upregulate epithelial-mesenchymal transition (EMT). The purpose of this study was to assess the effect of anti-inflammatory drugs on EMT pathogenesis in vitro and of cigarette smoke exposure on EMT markers in vivo.
Methods :
Human ARPE-19 cells were pre-treated with a direct TNF-α inhibitor (Cas 1049741) or Bay-11, an NF-κB inhibitor, before culture with concentrations of cigarette smoke extract (CSE) ranging from 0% to 1%. Cells were harvested after 24 hours and analyzed by quantitative PCR (qPCR) for known markers of inflammation and EMT, with at least 3 replications. In vivo mouse eyes were injected with ARPE-19 cells alone or ARPE-19 cells treated and resuspended in 0.5% CSE and sacrificed at 4 weeks for histologic analysis and immunohistochemistry staining (n=25). T-test and qualitative analysis were performed for in vitro and in vivo assays, respectively.
Results :
ARPE-19 cells pre-treated with direct TNF-α inhibitor showed no significant differences in TNF-α expression but significantly decreased expressions of Snail (by 53%), IL-6 (by 50%), and IL-8 (by 82%) mRNA (p<0.05). Bay-11 pre-treatment resulted in 78% decreased TNF-α as well as decreased expressions in Snail (by 77%), IL-6 (by 74%), and IL-8 (by 93%) (p<0.05). Our in vivo studies showed a significantly higher mean PVR grade in mice injected with CSE-treated RPE cells (4.82 +/- 1.08) compared to mice injected with RPE cells alone (3.43 +/- 1.45, p<0.05). Immunohistochemistry data showed that intravitreal injection of CSE-treated RPE resulted in significantly elevated vimentin and α-SMA protein expression in mouse retinal tissue, compared to RPE cells alone.
Conclusions :
We demonstrate that inhibiting TNF-α or the NF-κB pathway in RPE cells exposed to CSE also inhibits downstream proinflammatory and EMT pathways, suggesting that the TNF-α/NF-κB/Snail pathway may be a viable target for inhibiting EMT pathogenesis and PVR progression.
This is a 2021 ARVO Annual Meeting abstract.