Ts were seen after exposure to 8 of CSE. In these cells, the Apo J mRNA expression increased by 2.9+/20.3 fold (Fig. 5A), the CTGF expression by 4.8+/20.6 fold (Fig. 5B), and the fibronectin expression by 3.5+/ 20.6 fold (Fig. 5C), as compared to untreated control cells.Cigarette smoke extract induced protein expression of Apo J and CTGFThe protein expression of Apo J and CTGF was analysed by western blot analysis. Data are expressed as x-fold changes compared to the signals of untreated control cells (Figure 6). Protein expressions of Apo J and CTGF were measured after 223488-57-1 site treatment with 2, 4, and 8 of CSE. 1531364 There was a marked increase of Apo J protein expression after treatment of cultured human RPE cells with 4 and 8 of CSE as compared to untreated control cells (2 CSE: 1.060.1 fold; 4 CSE: 1.860.1 fold; 8 CSE: 2.260.8 fold) (Figure 6A). Similarly, CTGF protein expression was significantly elevated after exposure to 4 and 8 of CSE compared to untreated control cells (2 CSE: 1.160.5 fold; 4 CSE: 1.660.3 fold; 8 CSE: 2.060.6 fold) (Figure 6B).Cigarette smoke extract induced fibronectin and laminin secretionTo determine the fibronectin and laminin secretion of cultured human RPE cells by CSE exposure, we have used commercially available ELISA assays. Data are expressed as x-fold changes compared to the basal secretion levels of untreated control cells (Figure 7). Treatment of human RPE cells with 2, 4 and 8 of CSE increased the fibronectin secretion by 1.160.1 fold, 1.160.1 fold and 1.660.2 fold, as compared to untreated control cells. Furthermore, exposure of RPE cells to 2, 4 and 8 of CSE also led to increased levels of laminin secretion by 1.460.3 fold, 1.660.4 fold and 1.660.2 fold, compared to untreated control cells (Figure 7).DiscussionPrevious epidemiological studies have demonstrated that cigarette smoking significantly A-196 increases the risk of age-related macular degeneration (AMD) [7,8,9]. However, the impact of cigarette smoke on pathogenic processes of AMD is still unknown. One reason for the harmful effects of cigarette smoke on human cells is the generation of reactive oxygen species (ROS) and therefore oxidative stress [10]. Oxidative stress is also an important risk factor for ocular age-related diseases such as AMD. The loss of retinal pigment epithelial (RPE) cells is the major characteristic event of the atrophic form of AMD [39]. Previous in vitro studies have already demonstrated cytotoxic effects of cigarette smoke [40,41]. Cigarette smoke is known to contain an abundant number of toxic compounds. In ARPE-19 cells, specific toxic elements of cigarette smoke such as acrolein and benzopyrene may lead to reduced cell viability [40,41]. Cadmium, which is found in higher amounts in retinal tissues of AMD eyes, is 24786787 also released from cigarette smoke and can induce RPE cell death [42]. In our experiments, treatment of primary human RPE cells with 2, 4, and 8 of cigarette smoke extract (CSE) had no significant effects onFigure 5. CSE increased Apo J, CTGF, fibronectin mRNA expression. mRNA expression of (A) Apo J, (B) CTGF, (C) fibronectin. Real-time PCR analysis was conducted after treatment with 2, 4, and 8 of CSE. Results were normalized to GAPDH as reference. The steadystate mRNA levels of these senescence-associated genes in untreated control cells were set to 100 . Results are given as mean 6 s.d. of nine experiments with three different cell cultures from different donors (*P,0.05). Co, control. doi:10.1371/jo.Ts were seen after exposure to 8 of CSE. In these cells, the Apo J mRNA expression increased by 2.9+/20.3 fold (Fig. 5A), the CTGF expression by 4.8+/20.6 fold (Fig. 5B), and the fibronectin expression by 3.5+/ 20.6 fold (Fig. 5C), as compared to untreated control cells.Cigarette smoke extract induced protein expression of Apo J and CTGFThe protein expression of Apo J and CTGF was analysed by western blot analysis. Data are expressed as x-fold changes compared to the signals of untreated control cells (Figure 6). Protein expressions of Apo J and CTGF were measured after treatment with 2, 4, and 8 of CSE. 1531364 There was a marked increase of Apo J protein expression after treatment of cultured human RPE cells with 4 and 8 of CSE as compared to untreated control cells (2 CSE: 1.060.1 fold; 4 CSE: 1.860.1 fold; 8 CSE: 2.260.8 fold) (Figure 6A). Similarly, CTGF protein expression was significantly elevated after exposure to 4 and 8 of CSE compared to untreated control cells (2 CSE: 1.160.5 fold; 4 CSE: 1.660.3 fold; 8 CSE: 2.060.6 fold) (Figure 6B).Cigarette smoke extract induced fibronectin and laminin secretionTo determine the fibronectin and laminin secretion of cultured human RPE cells by CSE exposure, we have used commercially available ELISA assays. Data are expressed as x-fold changes compared to the basal secretion levels of untreated control cells (Figure 7). Treatment of human RPE cells with 2, 4 and 8 of CSE increased the fibronectin secretion by 1.160.1 fold, 1.160.1 fold and 1.660.2 fold, as compared to untreated control cells. Furthermore, exposure of RPE cells to 2, 4 and 8 of CSE also led to increased levels of laminin secretion by 1.460.3 fold, 1.660.4 fold and 1.660.2 fold, compared to untreated control cells (Figure 7).DiscussionPrevious epidemiological studies have demonstrated that cigarette smoking significantly increases the risk of age-related macular degeneration (AMD) [7,8,9]. However, the impact of cigarette smoke on pathogenic processes of AMD is still unknown. One reason for the harmful effects of cigarette smoke on human cells is the generation of reactive oxygen species (ROS) and therefore oxidative stress [10]. Oxidative stress is also an important risk factor for ocular age-related diseases such as AMD. The loss of retinal pigment epithelial (RPE) cells is the major characteristic event of the atrophic form of AMD [39]. Previous in vitro studies have already demonstrated cytotoxic effects of cigarette smoke [40,41]. Cigarette smoke is known to contain an abundant number of toxic compounds. In ARPE-19 cells, specific toxic elements of cigarette smoke such as acrolein and benzopyrene may lead to reduced cell viability [40,41]. Cadmium, which is found in higher amounts in retinal tissues of AMD eyes, is 24786787 also released from cigarette smoke and can induce RPE cell death [42]. In our experiments, treatment of primary human RPE cells with 2, 4, and 8 of cigarette smoke extract (CSE) had no significant effects onFigure 5. CSE increased Apo J, CTGF, fibronectin mRNA expression. mRNA expression of (A) Apo J, (B) CTGF, (C) fibronectin. Real-time PCR analysis was conducted after treatment with 2, 4, and 8 of CSE. Results were normalized to GAPDH as reference. The steadystate mRNA levels of these senescence-associated genes in untreated control cells were set to 100 . Results are given as mean 6 s.d. of nine experiments with three different cell cultures from different donors (*P,0.05). Co, control. doi:10.1371/jo.