A possible role of acrolein in diabetic retinopathy: involvement of a VEGF/TGFβ signaling pathway of the retinal pigment epithelium in hyperglycemia

Abstract

Purpose: Acrolein has been implicated in retinal pigment epithelium (RPE) cell death, and has been associated with diabetic retinopathy. Our purpose was to investigate the potential effect of high glucose in influencing acrolein-mediated RPE cytokine production and cell death. We investigated the influence of the acrolein effect on ARPE-19 cells in high glucose conditions and quantified the release of transforming growth factor β (TGFβ1 and 2) and vascular endothelial growth factor (VEGF). We assessed the ability of N-benzylhydroxylamine(NBHA) as well as TGFβ pathway inhibitors SIS3 and SB431542 to prevent this effect of acrolein on ARPE-19 cells.

Materials and methods: Confluent ARPE-19 cells were treated with acrolein and/or NBHA in both 5.5 and 18.8 mM glucose conditions. Cells were also pretreated with SIS3, a specific inhibitor of the SMAD3 pathway, and SB431542, a specific inhibitor of TGFβ signaling pathway, before treating them with acrolein. Viable cells were counted and ELISAs were performed to measure the cytokines TGFβ1 and 2, and VEGF released into the conditioned media.

Results: In ARPE-19 cells exposed to acrolein and hyperglycemia there was reduced cell viability and an increase in the cell media of VEGF, TGFβ1, and TGFβ2, which was reversed by NBHA. Acrolein/hyperglycemia-induced cell viability reduction and cytokine overproduction was also reduced by TGFβ pathway blockade.

Conclusions: We conclude that the effect of acrolein on the reduction of viability and VEGF increase by ARPE-19 cells in hyperglycemic media is conducted through the TGFβ signaling pathway. Our results suggest that benefits of sequestering acrolein by NBHA and the blockage of the TGFβ pathway by SB431542 and SIS3 offer suggestions as to potential useful pharmacological drug candidates for the prevention of diabetes-induced complications in the eye.

FIGURE 1

High glucose-enhances acrolein effects on ARPE-19 cell viability and VEGF levels. Confluent ARPE-19 cells were treated with either 5.5 or 18 mM glucose in combination with either 0, 12.5, 25, or 50 µM acrolein for 72 hours. Viable cell numbers (A) or VEGF (B) was determined for each treatment group. (A) 18 mM glucose increased cell loss at the 25 and 50 µM level of acrolein compared to cells treated with 5.5 mM glucose and an equivalent acrolein level, p < 0.05, p < 0.01. Two-way ANOVA was significant for the synergistic effect of glucose and acrolein on the loss of cell viability, p < 0.05. (B) 18 mM glucose increased the level of VEGF secreted into the conditioned media at each level of acrolein compared to cells treated with 5.5 mM glucose and an equivalent acrolein level. This reached significance at the 25 and 50 µM acrolein levels, p < 0.01, p < 0.001. Two-way ANOVA was significant for the synergistic effect of glucose and acrolein on the increase of VEGF, p < 0.0001. Single asterisk indicates a significant difference between 5.5 and 18 mM glucose at the same acrolein concentration (n = 3).

FIGURE 2

NBHA inhibits the effects of acrolein. Confluent ARPE-19 cells were treated with either 5.5 or 18 mM glucose in combination with either no acrolein, 50 µM acrolein, 200 µM NBHA, or a combination of 50 µM acrolein with either 50, 100, or 200 µM NBHA for 72 hours. Viable cell numbers were counted (A) and from the cell media the proteins VEGF (B), TGFβ2 (C), and TGFβ1 (D) were determined per 10,000 cells. Protein levels were divided by their corresponding cell count. (A) 200 µM of NBHA when used alone in 18 mM glucose decreased viable cell numbers by 25% compared to cell numbers from cells with no NBHA or acrolein treatment, p < 0.05. 50 µM acrolein used with 50 µM NBHA increased viable cell numbers by 73%, 100 µM NBHA increased by 103% and 200 µM NBHA by 186%, p < 0.001 compared to acrolein alone. A treatment effect was found by two-way ANOVA for the NBHA on acrolein-induced reduction in cell number, p < 0.0001. Results are average viable cell number ± SE, n = 3. (B) In combination with 50 µM acrolein in 18 mM glucose, 100 µM NBHA reduced VEGF by 44%, p < 0.001 and 200 µM NBHA reduced VEGF by 56%, p < 0.001, bringing it to within 4% of the nonacrolein, non-NBHA group. Results are the mean pg VEGF per 10,000 viable cells ± SE, n = 3. (C) Treatment with 50 µM acrolein and 18mM glucose resulted in 230%, p < 0.001, more TGFβ1 in the condition media compared to the corresponding 18 mM glucose group without acrolein. Addition of 200 µM NBHA to the treating media eliminated this effect. Two-way ANOVA found a significant correlation between the acrolein/NBHA group and glucose levels, p < 0.0001. Single asterisk indicates a significant difference within the same glucose level; “#” indicates a significant difference between glucose levels with the same treatment. Results are the mean pg TGFβ1 per 10,000 viable cells ± SE (n = 3). (D) In the 18 mM glucose groups, 50 µM of acrolein resulted in a 75% increase, p < 0.05, of TGFβ2 levels per 10,000 cells which was reduced to control with a combination of 200 µM NBHA to the treatment media. Asterisk refers to a significant difference between acrolein effect within its own glucose level. Results are the mean pg TGFβ2 per 10,000 viable cells ± SE (n = 3).

FIGURE 3

SIS3 and SB431542 inhibits the effects of acrolein effects in high glucose. Treatment with the Smad3 inhibitor SIS3 or the TGFβ1 receptor blocker SB431542 was performed on confluent ARPE-19 cells before they were treated with acrolein in both low (5.5 mM glucose), or high (18 mM glucose) for 72 hours. (A) Pretreatment with SB431542 and SIS3 prevented acrolein-induced loss of cell viability in both 5.5 and 18 mM glucose. These blockers were equally effective in preventing 50 µM acrolein-induced viable cell loss and resulted in treated cell numbers equivalent to those without acrolein treatment in both glucose groups. Results are average viable cell number ± SE (n =3). (B) Pretreatment with SB431542 and SIS3 prevent acrolein-induced VEGF increases per 10,000 cells in the 5.5 and 18 mM glucose/50 µM acrolein treatment groups. These blockers were equally effective in preventing VEGF increase and resulted in VEGF per 10,000 cells found in the condition media equivalent to that without acrolein treatment in both glucose groups. ELISA for VEGF was performed on the conditioned media from each well, and the results were divided by the corresponding viable cell counts. Results are the mean pg VEGF per 10,000 viable cells ± SE (n =3).