Fig 1.
Cigarette smoke extract (CSE) increases TNFα and EMT markers in ARPE-19 cells.
(A) ARPE-19 cells were treated with untreated culture medium (0% CSE) or 0.5% or 1% CSE in culture medium for 4 or 24 hours. After treatment, cells were collected and RNA was isolated. Exposure to 0.5 or 1% CSE for 4 or 24 hours induced expression of TNFα (left) and Snail (right) mRNA. mRNA levels are normalized to GAPDH mRNA levels and presented as fold change over untreated cells(0% CSE). Error bars represent standard error of the mean, with N = 3 for each point. * = p<0.05 and ** = p<0.01 compared to 0% CSE. (B) Representative cell culture images of ARPE-19 cells without CSE (0% CSE) or with 0.5 or 1% CSE at 4 and 24 hours. CSE alters the morphology of the ARPE-19 cells to become more rounded, especially at 24 hours treatment. (C) ARPE cells were treated with or without CSE as in (A) for 24 hours. Afterwards, cells were lysed and protein expression was analyzed by Western blot. CSE elevates both αSMA and SNAIL levels. (D) ARPE-19 cells were treated with 1% CSE in the presence or absence of 1 uM of the NF-κB inhibitor, BAY-11-7082 (BAY). After 24 hours, cells were harvested and protein and mRNA levels were analyzed as described above. Inhibition of NF-κB mitigated the ability of CSE to induce SNAIL and EMT marker, vimentin. (E) RNA was isolated from cells treated as in (D). BAY-11-7082 mitigated the ability of CSE to induce TNFα mRNA.
Fig 2.
Inhibition of the TNFα-dependent NF-κB pathway blocks SNAIL and inflammatory cytokine production in CSE exposed ARPE-19 cells.
ARPE-19 cells exposed to CSE for 24 hours in the presence or absence of the NF-κB inhibitor, BAY-11-7082 (BAY, 1 μM) or the TNFα inhibitor, CAS 1049741 (CAS, 1 μM). After exposure, total RNA was isolated and analyzed by qPCR. CSE exposed ARPE-19 cells produced elevated SNAIL (A), IL6 (B), and IL8 (C) mRNA. Fold differences were normalized to 0% CSE for each condition. (D) TNFα and (E) IL-6 production from culture supernatants from cells treated as above were analyzed by ELISA. CSE exposure increased both TNFα and IL-6. Inhibition of NF-κB with BAY decreased TNFα production in ARPE-19 cells cultured with CSE while CAS did not. Both BAY and CAS significantly decreased IL-6 production. (F) APRE-19 cells were treated with control, TNFα or RELA siRNA for 24 hours. After treatment with siRNA, cells were exposed, or not, to 1.0% CSE for 24 hours. After exposure, cells were harvested and RNA levels were analyzed by qPCR. SNAIL mRNA levels were significantly increase by CSE exposure in control siRNA treated cells while SNAIL mRNA production was significantly attenuated in cells cultured with TNFα or RELA siRNA. Error bars represent standard error of data set. N≥3 for each. *p<0.05, **p<0.01, ***p<0.001.
Fig 3.
Intravitreal injection in mice with CSE-treated RPE cells resulted in more rapid and severe PVR development.
RPE only injections consisted of 2,000 ARPE-19 cells resuspended in 1uL media (N = 14). CSE-treated RPE injections consisted of 2,000 ARPE-19 cells cultured and resuspended in 0.5% CSE (N = 11). PVR grades are based on fundus photography. (A) Both RPE only and CSE-treated RPE injected eyes developed more severe PVR over time, although CSE-treated RPE injected mice developed significantly worse PVR at every timepoint. (B) Representative fundus images of eyes injected with CSE only, RPE only, or CSE-treated RPE at each timepoint. Assigned PVR grade (PG) is listed bottom right of each image. (C) Table comparing mean PVR grades of eyes injected with RPE only or CSE-treated RPE cells.
Fig 4.
CSE treated RPE cell injection increased retinal folds, retinal detachment and PVR membranes thickness compared to control RPE.
Fundus imaging, OCT, and histology of representative eyes from mice injected intravitreally with control RPE cells (A,B,C) vs. RPE cells treated and resuspended with 0.5% CSE (D,E,F). All images were taken at 4 weeks post-RPE injection. Intravitreal injection of CSE-treated RPE resulted in development of more severe PVR by 4 weeks with evidence of retinal folding, significant areas of detachment, and inflammatory infiltrate. The inflammatory infiltrate is especially prevalent in the vitreous of the top section in F. Arrows and * in E and F highlight retinal folds observed on OCT imaging seen in histologic cross-sections. Scale bars represent 100 microns (μm) in length.
Fig 5.
CSE treated RPE cells increased expression of Vimentin and αSMA in PVR compared to control RPE cells.
Immunohistochemistry sections of representative eyes from mice injected intravitreally with control RPE cells vs. RPE cells treated and resuspended with 0.5% CSE. Like in Fig 4, images were taken at 4 weeks post-RPE injection. Intravitreal injection of CSE-treated RPE resulted in significantly elevated levels of vimentin and αSMA protein expression (brown pigment) in both vitreous and retinal tissue. The arrows and * highlight increases in the amount of cellularity and in the amount of vimentin and αSMA staining in and near the retina in CSE-treated samples. The scale bar at the bottom right represents 100 microns (μm) in length.
Fig 6.
A model of cigarette smoke mechanism of action on RPE cells.
Cigarette smoke stimulates production of TNF-α intracellularly, which activates IκB kinase, phosphorylating IκB. This results in dissociation of IκB from NF-κB, which enters the nucleus to activate transcription of Snail, IL-6, and IL-8. Snail triggers downstream factors involved in epithelial-mesenchymal transition.