Urnal.pone.0048501.gRPE cell loss. However, exposure of cells to 12 of

Urnal.pone.0048501.gRPE cell loss. However, exposure of cells to 12 of CSE markedly induced RPE cell death. At the first glance, these results are in contrast to previous investigations with ARPE-19 cells, which showed a decreased Dimethyloxallyl Glycine chemical information viability after 0.5 of CSE [43]. However, it must be taken into account that in Bertram et al. [43], CSE was generated by the smoke of research-grade cigarettes (Kentucky Tobacco Research Council, Lexington, KY, U.S.A.), which contain a much higher nicotine concentration than commercially available filter cigarettes. Therefore, CSE may be toxic for RPEEffects of Smoke in RPEFigure 6. CSE increased Apo J, CTGF protein expression. Protein expression of (A) Apo J, (B) CTGF. Data are expressed as x-fold changes compared to the signals of untreated control cells and represent the mean 6 s.d. 23115181 of results of three experiments with three different cell cultures from different donors (*P,0.05). doi:10.1371/journal.pone.0048501.gcells at higher concentrations. Interestingly, Patil et al. [44] did not find decreased cell viability of human ARPE-19 cells after treatment with nicotine itself. This observation may be explained by the fact that not only nicotine itself but also other toxic elements of cigarette smoke influence the RPE viability. Furthermore, in our subsequent experiments, treatment of primary human RPE cells with 2, 4, and 8 of CSE increased lipid peroxidationestimated by the loss of cis-parinaric acid (PNA) fluorescence. These results suggest that lower Dovitinib (lactate) concentrations of CSE can induce the release of ROS and thus cause oxidative stress in primary human RPE cells. At the cellular level, oxidative stress can trigger the so-called `stress-induced premature senescence’ (SIPS) [15,45]. There is a growing body of evidence suggesting that RPE cells also undergoFigure 7. CSE increased fibronectin, laminin protein secretion. Protein secretion of (A) fibronectin (FN) and (B) laminin into culture media. Error bars: 6 s.d. of results from three experiments with three different cell cultures (*P,0.05). Co, control. doi:10.1371/journal.pone.0048501.gEffects of Smoke in RPEan accelerated ageing process in AMD [24,46,47,48]. We have previously shown that sublethal concentrations of hydrogen peroxide induced senescence-associated ?Galactosidase (SA- al) activity in primary cultured RPE cells [29]. In the experiments of the current study, treatment of primary human RPE cultures with CSE could significantly increase the proportion of SA-?Gal positive cells. Positive staining of SA-?Gal has also been detected in vitro in late passage RPE cultures [49,50] and in 1662274 vivo in the RPE cells of old primate eyes [51]. In human RPE cells, an increased expression of SA-?Gal staining could be triggered by mild hyperoxia-mediated ROS release [52]. Furthermore, cellular senescence can also be identified by increased expression of senescence-associated biomarkers such as Apo J, CTGF, and fibronectin. All three biomarkers are inducible by oxidative stress [16,18]. In our experiments, exposure of primary human RPE cells to CSE could lead to a significant elevation of Apo J, CTGF, and fibronectin expression. The cellular chaperone Apo J has been previously detected in the RPE of AMD donor eyes, although its role and function in the RPE is still unclear [19,53]. In contrast, CTGF and fibronectin have been found in the Bruch’s membrane, in drusen and in basal linear deposits of AMD eyes [20,21,22]. Furthermore, we could show that treatment of hu.Urnal.pone.0048501.gRPE cell loss. However, exposure of cells to 12 of CSE markedly induced RPE cell death. At the first glance, these results are in contrast to previous investigations with ARPE-19 cells, which showed a decreased viability after 0.5 of CSE [43]. However, it must be taken into account that in Bertram et al. [43], CSE was generated by the smoke of research-grade cigarettes (Kentucky Tobacco Research Council, Lexington, KY, U.S.A.), which contain a much higher nicotine concentration than commercially available filter cigarettes. Therefore, CSE may be toxic for RPEEffects of Smoke in RPEFigure 6. CSE increased Apo J, CTGF protein expression. Protein expression of (A) Apo J, (B) CTGF. Data are expressed as x-fold changes compared to the signals of untreated control cells and represent the mean 6 s.d. 23115181 of results of three experiments with three different cell cultures from different donors (*P,0.05). doi:10.1371/journal.pone.0048501.gcells at higher concentrations. Interestingly, Patil et al. [44] did not find decreased cell viability of human ARPE-19 cells after treatment with nicotine itself. This observation may be explained by the fact that not only nicotine itself but also other toxic elements of cigarette smoke influence the RPE viability. Furthermore, in our subsequent experiments, treatment of primary human RPE cells with 2, 4, and 8 of CSE increased lipid peroxidationestimated by the loss of cis-parinaric acid (PNA) fluorescence. These results suggest that lower concentrations of CSE can induce the release of ROS and thus cause oxidative stress in primary human RPE cells. At the cellular level, oxidative stress can trigger the so-called `stress-induced premature senescence’ (SIPS) [15,45]. There is a growing body of evidence suggesting that RPE cells also undergoFigure 7. CSE increased fibronectin, laminin protein secretion. Protein secretion of (A) fibronectin (FN) and (B) laminin into culture media. Error bars: 6 s.d. of results from three experiments with three different cell cultures (*P,0.05). Co, control. doi:10.1371/journal.pone.0048501.gEffects of Smoke in RPEan accelerated ageing process in AMD [24,46,47,48]. We have previously shown that sublethal concentrations of hydrogen peroxide induced senescence-associated ?Galactosidase (SA- al) activity in primary cultured RPE cells [29]. In the experiments of the current study, treatment of primary human RPE cultures with CSE could significantly increase the proportion of SA-?Gal positive cells. Positive staining of SA-?Gal has also been detected in vitro in late passage RPE cultures [49,50] and in 1662274 vivo in the RPE cells of old primate eyes [51]. In human RPE cells, an increased expression of SA-?Gal staining could be triggered by mild hyperoxia-mediated ROS release [52]. Furthermore, cellular senescence can also be identified by increased expression of senescence-associated biomarkers such as Apo J, CTGF, and fibronectin. All three biomarkers are inducible by oxidative stress [16,18]. In our experiments, exposure of primary human RPE cells to CSE could lead to a significant elevation of Apo J, CTGF, and fibronectin expression. The cellular chaperone Apo J has been previously detected in the RPE of AMD donor eyes, although its role and function in the RPE is still unclear [19,53]. In contrast, CTGF and fibronectin have been found in the Bruch’s membrane, in drusen and in basal linear deposits of AMD eyes [20,21,22]. Furthermore, we could show that treatment of hu.